Medicinal composition of amaranth extract having enriched nitrate content and a method of preparing the same

ABSTRACT

An extract of Amaranth, having enriched nitrate content, L-arginine, Flavonoids, saponins, alkaloids, carbohydrates, proteins, potassium, and with negligible amount of oxalic acid or oxalate content. The extract of Amaranth can be prepared from fresh or dried leaves and stem of Amaranth. Dosage forms of extract include fast melt tablet, lozenge, candy, chewing gum, beverage, tablet, capsule, pill, and powder. The extract of Amaranth enhances nitric oxide concentration, enhances nitrate level concentration, and enhances nitrite level in blood as well as in saliva. Administering the extract can lower blood pressure, increase physical endurance, increase swimming endurance, increase running endurance, and improve sexual performance among human beings.

This application is a continuation of PCT International ApplicationSerial Number PCT/IN2014/000430, filed Jun. 26, 2014, which claimspriority of Indian Provisional Application No. filed 2861/CHE/2013,filed Jun. 28, 2013, all of which are incorporated by reference.

BACKGROUND

Nitric oxide is an important cellular signaling molecule involved inmany physiological and pathological processes. It is a powerfulvasodilator with a short half-life of a few seconds in the blood. NitricOxide is a free form of gas produced by the cells in our body. It isproduced when enzymes in the body break down the amino acid Arginine,and is mainly used for intra-cellular communication. Nitric Oxide isrequired for key physiological functions within our body. It travelsfreely from one cell to another assisting in a variety of biologicalfunctions. Depending on the requirement, Nitric Oxide can act as ahormone, a neurotransmitter and an intracellular messenger.

Nitric Oxide is crucial for a variety of body functions, and it isessential that the cells produce adequate amount of this gas within ourbodies. Body builders or those who perform rigorous physical activitiesneed a higher amount of NO, as it allows for an increase in blood flowwhile building muscles. Signs of NO deficiency include extreme fatigueand physical weakness. It is useful in treating a variety of conditionssuch as insomnia, obesity, diabetes and sexual problems. In thecardiovascular system, NO is an important determinant of basal vasculartone, prevents platelet activation, limits leukocyte adhesion to theendothelium, and regulates myocardial contractility. NO may also play arole in the pathogenesis of common cardiovascular disorders, includinghypotension accompanying shock states, essential hypertension, andatherosclerosis. Nitric oxide regulates blood circulation throughout thebody, increases the diameter of blood vessels and prevents formation ofclots. It assists the endothelial cells in controlling and relaxingblood vessels. Nitric oxide is a cytotoxic agent of macrophages, amessenger molecule of neurons, and a vasodilator produced by endothelialcells. The Immune cells within our body release Nitric Oxide to destroybacteria, virus and other harmful foreign organisms that can causeinfections. NO is also known to prevent tumour and cancerous growthswithin the body cells.

Nitric Oxide has been shown to be mediator of erectile function. NitricOxide stimulates, invigorates and amplifies the sexual responsemechanism within the body. Sensory and mental stimulation causes thenerve cells to release nitric oxide. This causes the penis muscles torelax, allowing blood to flow into the penis and create an erection. Theprocess remains the same for women too, as blood flow increases in theirvaginal tissues. Nitric Oxide increases the endurance level of themuscle cells, enabling you to lift heavier load and perform strenuousactivities with ease. Nitric Oxide acts as an intracellular messengerbetween various cells in the body, including the nerve cells. Withadequate amount of NO present in the body, the communication betweennerve cells is faster, leading to quick responses and an increase infocus and vigilance. Common disorders that promote atherosclerosis, suchas hypertension, hyperlipidemia, smoking, and diabetes, are allassociated with abnormal endothelial function, one manifestation ofwhich is a comparative deficiency of bioactive NO. A deficiency of NOproducing neurons in the gastrointestinal tract is believed to beresponsible for certain abnormalities in gastrointestinal motility, suchas Hirschsprung's disease, achalasia, and chronic intestinalpseudo-obstruction. NO is also believed to play an important role ingastric cytoprotection, possibly by way of increased mucosal blood flowand the modulation of gastric epithelial function.

Production of Nitric Oxide in Human Body (Biosynthesis)

NO is produced from the amino acid L-arginine by the enzymatic action ofnitric oxide synthase (NOS). There are two endothelial forms of NOS:constitutive NOS (cNOS; type III) and inducible NOS (iNOS; type II).Co-factors for NOS include oxygen, NADPH, tetrahydrobiopterin and flavinadenine nucleotides. In addition to endothelial NOS, there is a neuralNOS (nNOS; type I) that serves as a transmitter in the brain and indifferent nerves of the peripheral nervous system, such asnon-adrenergic, non-cholinergic autonomic nerves that innervate penileerectile tissues and other specialized tissues in the body to producevasodilation.

Under normal, basal condition in blood vessels, NO is continually beingproduced by cNOS. The activity of cNOS is calcium andcalmodulin-dependent. There are two basic pathways for the stimulationof cNOS, both of which involve release of calcium ions fromsubsarcolemmal storage sites. First, shearing forces acting on thevascular endothelium generated by blood flow causes a release of calciumand subsequent cNOS activation. Therefore, increases in blood flowstimulate NO formation (flow-dependent NO formation). Second,endothelial receptors for a variety of ligands stimulate calcium releaseand subsequent NO production (receptor-stimulated NO formation).Included are receptors for acetylcholine, bradykinin, substance-P,adenosine, and many others vasoactive substances.

The other isoform of endothelial NOS is iNOS. It differs, in part, fromcNOS in that its activation is calcium independent. Under normal, basalconditions, the activity of iNOS is very low. The activity of iNOS isstimulated during inflammation by bacterial endotoxins (e.g.,lipopolysaccharide) and cytokines such as tumor necrosis factor (TNF)and interleukins. During inflammation, the amount of NO produced by iNOSmay be a 1,000-fold greater than that produced by cNOS.

Production of NO from Dietary Nitrates

Dietary nitrate is an important source of nitric oxide in mammals.Green, leafy vegetables, and some root vegetables (such as beetroot)have high concentrations of nitrate. When eaten and absorbed into thebloodstream nitrate is concentrated in saliva (about 10 fold) and isreduced to nitrite on the surface of the tongue by a biofilm ofcommensal facultative anaerobic bacteria. This nitrite is swallowed andreacts with acid and reducing substances in the stomach (such asascorbate) to produce high concentrations of nitric oxide.

An Acceptable Daily Intake (ADI) for nitrate of 3.7 mg/kg b.w./day,equivalent to 222 mg nitrate per day for a 60 kg adult was establishedby the former Scientific Committee on Food (SCF) and was reconfirmed bythe Joint FAO/WHO Expert Committee on Food Additives (JECFA).

The bioactivation of nitrate from dietary or endogenous sources requiresits initial reduction to nitrite, and because mammals lack specific andeffective nitrate reductase enzymes, this conversion is mainly carriedout by commensal bacteria in the gastrointestinal tract and on bodysurfaces. Nitrite is unique to the nitrogen oxides in its redox positionbetween oxidative (NO₂ radical) and reductive (NO radical) signallingand its relative stability in blood and tissue. Once nitrite is formed,there are numerous pathways in the body for its further reduction to NO,involving haemoglobin, myoglobin, xanthine oxidoreductase, ascorbate,polyphenols and protons. The generation of NO by these pathways isgreatly enhanced during hypoxia and acidosis, thereby ensuring NOproduction in situations for which the oxygen-dependent NOS enzymeactivities are compromised. Dietary nitrate is rapidly absorbed in theupper gastrointestinal tract. In the blood, it mixes with the nitrateformed from the oxidation of endogenous NO produced from the NOSenzymes. After a meal rich in nitrate, the levels in plasma increasegreatly and remain high for a prolonged period of time.

Once in the oral cavity, commensal facultative anaerobic bacteria usenitrate as an alternative electron acceptor to oxygen duringrespiration, effectively reducing salivary nitrate to nitrite by theaction of nitrate reductases. Human nitrate reduction requires thepresence of these bacteria—suggesting a functional symbiosisrelationship—as mammalian cells cannot effectively metabolize thisanion. The salivary nitrate levels can approach 10 mM and nitrite levels1-2 mM after a dietary nitrate load. When saliva enters the acidicstomach (1-1.5 1 per day), much of the nitrite is rapidly protonated toform nitrous acid (HNO₂; pKa ˜3.3), which decomposes further to form NOand other nitrogen oxides. Nitrite reduction to NO is greatly enhancedby reducing compounds such as vitamin C and polyphenols, both of whichare abundant in the diet.

The percentage of nitrate content in leafy vegetables is very less tosignificantly increase the nitric oxide level in the blood when takenorally. A larger quantity is required to be ingested at regular intervalto get significant level of nitric oxide in the blood. Consumption oflarge quantity of leafy vegetables on regular basis is not convenient insuch a busy life. Thus, providing a supplement developed from nitratecontaining plant in the form of compact delivery system will be aconvenient alternate to generate higher nitric oxide content in theblood. Moreover, as nitrates are required to be converted into nitritesin the mouth itself (by commensal facultative anaerobic bacteria), itwill be more advantageous if the nitrate rich formulation can bedelivered to oral cavity in the form of chewing gum, lozenge, candy etc.Delivery via oral cavity offers certain advantages over conventionaldelivery of drugs.

The sites of drug administration in the oral cavity include the floor ofthe mouth (sublingual), the inside of the cheeks (buccal) and the gums(gingival). Drug administration via oral cavity offers several distinctadvantages:

-   -   The buccal mucosa is relatively permeable with a rich blood        supply, robust in comparison to the other mucosal tissues.    -   Bypass the first-pass effect and non-exposure of the drugs to        the gastrointestinal fluids.    -   Improve the performance of many drugs, as they are having        prolonged contact time with the mucosa.    -   High patient acceptance compared to other non-oral routes of        drug administration.    -   As a result of adhesion and intimate contact, the formulation        stays longer at the delivery site improving API bioavailability        using lower API concentrations for disease treatment.    -   Harsh environmental factors that exist in oral delivery of a        drug are circumvented by buccal drug delivery.

A lot of work has been done for enhancing the level of nitric oxide inthe blood for human beings and method of administration ofmedicaments/formulations, including those originated from plants(botanical source) available in nature in plenty. A short review of someof the prior art will elucidate how we have addressed the issue comparedto other prior art and how effectively we could address enhancing thelevel of nitric oxide in the blood for human beings and the method oftreating diseases as well as source for overcoming certain deficienciesof humanbeings.

U.S. Pat. No. 8,303,995, Bryan et al, discloses a nitrite formulationsand their use as nitric oxide prodrugs. The Compositions comprising fromabout 40 weight parts to about 1000 weight parts of a botanical nitratesource; from about 20 weight parts to about 500 weight parts of abotanical source of nitrite reduction activity; and from about 4 weightparts to about 100 weight parts of a nitrite salt.

The botanical source of nitrite reduction activity is selected from thegroup consisting of hawthorn berry, Schisandra, green tea, beet root,pine bark, holy basil, gymnema sylvestre, ashwagandha root, salvia, St.John wort, broccoli, stevia, spinach, gingko, kelp, tribulus, eleuthero,epimedium, eucommia, rhodiola, green tea, codonopsys, panax ginseng,astragalus, dodder seed, cordyceps, berries, tea, beer, grapes, wine,olive oil, chocolate, cocoa, coffee, walnuts, peanuts, borojo,pomegranates, popcorn, yerba mate, and mixtures thereof. Methods ofreducing triglycerides or reducing C-reactive protein levels are alsoprovided.

U.S. Pat. No. 8,298,589, Bryan et al, is related to ‘Nitriteformulations and their use as nitric oxide prodrugs having a compositioncomprising nitrite salt (10 mg to 100 mg), nitrate salt (50 mg to 500mg), ascorbic acid (100 mg to 2000 mg) of sodium and potassium.

Patent publication No US 20130071494, Bryan et al, also discloses anitrite formulations and their use as nitric oxide prodrugs. Thecompositions comprise of from about 40 weight parts to about 1000 weightparts of a botanical nitrate source; from about 20 weight parts to about500 weight parts of a botanical source of nitrite reduction activity;and from about 4 weight parts to about 100 weight parts of a nitritesalt Use of said composition in methods of reducing triglycerides orreducing C-reactive protein levels are also disclosed here.

Patent publication No US 20120321724, and WO 201323217 Bryan et al,discloses a method of measuring in vivo nitric oxide and nitrite levelsin individuals by providing a salivary nitrite test substrate, testingsalivary nitrite levels with the test substrate, measuring nitritelevels detected in the testing; and correlating the measured nitritelevels with in vivo nitric oxide bio-availability.

In patent No. U.S. Pat. No. 8,435,570, Bryan et al, disclose acomposition comprising a nitrite salt, a nitrate salt, and ascorbicacid. A method of enhancing cardiovascular performance or treatingadverse cardiovascular event in a mammal is also provided.

Patent publication No 20130071371, Bryan et al, is related to acomposition and method of providing nitric oxide and nitrite therapy topatients whereby a therapeutic amount including a dispersable medium isbioavailable within approximately 30 minutes of administrationincluding. In embodiments of the disclosure, nitric oxide is produced inthe oral cavity.

In the above patents, the nitrate source is either synthetic one or froma botanical nitrate source which includes spinach, beet root, artichoke,holybasil, green tea etc.

Supplementation of Amaranth extract enriched with nitrate enhances thenitrate and nitrite levels in the body. Presence of vitamin C promotesthe conversion of nitrite to nitric oxide in body thereby increasing theantioxidant potential in body.

The formulation of nitrate enriched Amaranth extract with Vitamin C inthe present formulation further improves the generation of nitric oxideby promoting the conversion of nitrite delivered from Amaranth extractto nitric oxide in the body. Vitamin C used may be of synthetic ornatural origin.

Amaranthus are dietary leafy vegetables belonging to the familyAmaranthaceae. Amaranth belongs to the genus Amaranthus and variousspecies include Amaranthus dubius, Amaranthus spinosus, Amaranthusfimbriatus, Amaranthus floridanus, Amaranthus graecizans etc. Otherdietary sources of nitrate are Spinach (Spinacia oleracia, family:Amaranthaceae), beet root (Beta vulgaris, family: Amaranthaceae),lettuce (Lactuca sativa, family: Asteraceae) etc.

The genus amaranthus contains approximately 70 species of worldwidedistribution including pigweeds, water hemps, and grain amaranthus. Forhuman consumption there are cultivated grain amaranths—A. caudatus, A.cruentus and A. hypochondriacus and vegetable amaranths—mainly A.dubius, A. tricolor and A. cruentus. Nowadays, the grain amaranthus arecultivated from the temperate to tropical zone and the vegetableamaranths mainly in the South Africa and South Asia.

These plants, which grow 90 centimeters to 150 centimeters tall, areabundant weeds in many parts of the world. All amaranth have alternatesimple leaves. They may have some red color present on the stems. Theybear minute, greenish flowers in dense clusters at the top of theplants. Their seeds may be brown or black in weedy species andlight-colored in domestic species. Some amaranth species have been grownas a grain crop and a garden vegetable in various parts of the world,especially in South America.

Amaranthus are very promising crops. The main reasons could be contentof protein, fat and active substances. The content of seed protein is inthe range 13-18% with very good balanced amino acids. The lysine contentis relatively high in the comparison with common cereals. The content ofcrude proteins in leaves is from 27 to 49% in d.m. what is more than inthe leaves in the spinach. Amaranthus have comparable or higher amountsof essential amino acids as whole egg protein. The fat content is in therange 0.8-8.0%. The linoleic acid is the predominant fatty acid, withlesser amount of oleic and palmitic acids.

SUMMARY

Disclosure provides a medicinal composition obtained from the extract ofAmaranth. Disclosure provides an extract of Amaranth having low oxalicacid content. The Amaranth extract includes

about 0.1% to about 20% nitrates;

about 0.1% to about 10% of L-arginine;

about 0.001% to about 10% of flavonoids;

about 0.01% to about 15% of saponins;

about 0.01% to about 10% of alkaloids;

about 10% to about 30% of carbohydrates;

about 5% to about 25% of proteins;

about 5% to about 25% potassium; and,

about 0.01% to about 50% of total oxalic acid. Determination of totaloxalic acid content included measurement of free oxalic acid andoxalates in the extract.

The disclosure also provides oral dosage forms of the composition ofAmaranth such as fast melt tablets, lozenge, chewing gum etc to make itconvenient for buccal absorption. The disclosure also provides dosageforms such as tablets, capsules, pills, powders etc for absorption fromthe stomach.

The disclosure provides methods for producing the Amaranth extract. Theextract of Amaranth can be prepared from fresh or from dried leaves andstem of Amaranth.

Some embodiments of the composition are found to be useful for theenhancing the amount of nitric oxide in blood plasma. Some embodimentsenhance the amount of nitric oxide in saliva. Some embodiments enhancethe level of nitrate and nitrite in blood plasma. Some embodiments ofthe extract enhance the level of nitrate and nitrite in saliva. Someembodiments enhance the antioxidant activity in mammals. In someembodiments, the composition lowers blood pressure (hypertension). Someembodiments of the composition increase endurance.

In some embodiments, the Amaranth extract can be fortified by addingamla extract.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Flow chart—Extraction of Fresh Amaranth with methanol.

FIG. 2: Flow chart—Extraction of Fresh Amaranth with water.

FIG. 3: Flow chart—Extraction of Dried Amaranth with methanol.

FIG. 4: Flow chart—Extraction of dried Amaranth with water.

FIG. 5: Flow chart—Extraction of Fresh Amaranth by treating withpectinase.

FIG. 6: Flow chart—Extraction of Fresh Amaranth by treating withpectinase and removal of Oxalic acid.

FIG. 7: Flow chart—Hexane Extract of Fresh Amaranth after treating withpectinase and removal of Oxalic acid.

FIG. 8: Flow chart—Chloroform Extract of Fresh Amaranth after treatingwith pectinase and removal of Oxalic acid.

FIG. 9: Flow chart—EDC Extract of Fresh Amaranth after treating withpectinase and removal of Oxalic acid.

FIG. 10: Flow chart—Extraction of Amla.

FIG. 11: Flow chart—Hexane Extract of Fresh Amaranth after treating withpectinase and removal of Oxalic acid in combination with Amla.

FIG. 12: Flow chart—Preparation of dried Amaranth leaf powder.

FIG. 13: Flow chart—Preparation of Amaranth Juice.

FIG. 14 (Part A): Table showing the percentage of actives in differentextract of Amaranth and fast dissolving tablets

FIG. 14 (Part B): Table showing the percentage of actives in differentextract of Amaranth and fast dissolving tablets

DETAILED DESCRIPTION

The disclosure provides an entirely different medicinal compositionprepared from Amaranth as nitrate source by a unique method ofextraction. The disclosed processes provide an extract of Amaranthenriched with nitrate, L-arginine, flavonoids, carbohydrate, protein,saponins, alkaloids, potassium and having negligible oxalic acid oroxalate content. Many vegetables contain high amount of total oxalicacid in the form of free oxalic acid or oxalate salts and the oxalic oroxalate content is toxic to human health. The extracts from vegetablescontain oxalic acid along with other active constituents. The extract ofAmaranth also contains oxalic acid and oxalates. But in the disclosedmethods, we removed the oxalic acid and oxalates from the Amaranthextract to provide an extract of Amaranth having negligible oxalic acidor oxalate content. The total oxalic acid content was measured by HPLCmethod and provides the total of both free oxalic acid and oxalate saltsin the extract.

The composition obtained from the enriched extract of amaranth is usefulfor lowering the blood pressure (hypertension), enhancing the level ofnitric oxide in the blood plasma and saliva, enhancing the level ofnitrate and nitrite in blood plasma and saliva, enhance the antioxidantactivity and increase the endurance. The disclosure relates to amedicinal composition of Amaranth extract. The composition has anenriched nitrate content, L-arginine, flavonoids, carbohydrate, protein,saponins, alkaloids, potassium and a negligible amount of oxalic acid oroxalate.

The Amaranth extract can be obtained from both fresh and dry leaves andstem of Amaranth. The extract when administered to mammals especially tohuman beings can lower the blood pressure (hypertension) and increasethe endurance. If the same extract is further enriched by enzymetreatment it can enhance the capability of lowering the blood pressure(hypertension) and increasing the endurance.

It is further noted that the composition of enriched Amaranth extractorigin has the property to enhance the amount of nitric oxide in bloodplasma and saliva as well as the property to enhance the level ofnitrate and nitrite in blood plasma and saliva. The composition is alsofound to be useful for enhancing the antioxidant activity.

Some embodiments provide a composition obtained from the extract ofAmaranth from fresh leaves and stem of Amaranth comprising enrichednitrate content, L-Arginine, flavonoids, saponins, alkaloids, potassiumand having negligible amount of oxalic acid or oxalate content whereinnitrates are present in the extract in a range of 3% to 20%, L-Arginineare present in the extract from 1% to 10%, flavonoids are present in theextract in a range of 1% and 10%, saponins are present in the range of5% to 15%, alkaloids are present in the range of 3% to 10%. The extractalso contains carbohydrate from 10% to 30%, potassium content from 5% to25% and proteins are present in a range of 5% to 25%.

Some embodiments provide an extract of Amaranth having low total oxalicacid content. The extract includes:

about 0.1% to about 20% nitrates;

about 0.1% to about 10% of L-arginine; (0.1-2%; 0.1-6%; 0.1-10%)

about 0.001% to about 10% of flavonoids;

about 0.01% to about 15% of saponins;

about 0.01% to about 10% of alkaloids; (0.01-5%)

about 10% to about 30% of carbohydrates;

about 5% to about 25% of proteins;

about 5% to about 25% potassium; and,

about 0.01% to about 50% of total oxalic acid. The total oxalic acidincludes free oxalic acid and oxalate salts in the extract.

In some embodiments of the extract, nitrates ranges from about 0.1% toabout 3%. In some embodiments of the extract, nitrates ranges from about1% to about 10%. In some embodiments of the extract, nitrates rangesfrom about 10% to about 20%. In some embodiments of the extract,nitrates ranges from about 3% to about 20%. In some embodiments of theextract, L-arginine ranges from about 0.1% to about 2%. In someembodiments of the extract, L-arginine ranges from about 0.1% to about6%. In some embodiments of the extract, L-arginine ranges from about0.1% to about 10%. In some embodiments of the extract, alkaloids rangesfrom about 0.01% to about 5%. In some embodiments of the extract, totaloxalic acid ranges from about 0.01% to about 0.1%. Total oxalic acidcontent in the extracts was determined by HPLC method to measure bothfree oxalic acid and oxalates content in the extracts. In someembodiments of the extract, total oxalic acid ranges from about 0.01% toabout 1%. In some embodiments of the extract, total oxalic acid rangesfrom about 0.1% to about 1%. In some embodiments of the extract, totaloxalic acid ranges from about 1% to about 10%. In some embodiments ofthe extract, total oxalic acid ranges from about 1% to about 20%. Insome embodiments of the extract, total oxalic acid ranges from about 1%to about 30%. In some embodiments of the extract, total oxalic acidranges from about 1% to about 40%. In some embodiments of the extract,total oxalic acid ranges from about 1% to about 50%. In some embodimentsof the extract, total oxalic acid ranges from about 10% to about 20%.

Some embodiments provide an extract of Amaranth having low total oxalicacid content. The extract includes

about 3% to about 20% nitrates;

about 1% to about 10% of L-arginine;

about 1% to about 10% of flavonoids;

about 5% to about 15% of saponins;

about 3% to about 10% of alkaloids;

about 10% to about 30% of carbohydrates;

about 5% to about 25% of proteins;

about 1% to about 5% of potassium; and,

about 0.1% total oxalic acid. Total oxalic acid was measured by HPLCmethod to determine free oxalic acid and oxalates in the extract.

The disclosure provides extracts having enriched nitrate, L-arginine,flavonoids, carbohydrate, protein, saponins, alkaloids, potassium andhaving negligible oxalic acid or oxalate content wherein the extractsmay be obtained from red Amaranth or Spleen amaranth or red Spinach(Amaranthus dubius) or any related species of amaranth like A. caudatus,A. cruentus, A. hypochondriacus, A. tricolor, A. blitum, A. viridus etc.

The disclosure also describes delivery of the nitrate enrichedformulations orally as fast melt tablets, lozenge, candy, chewing gum,beverage etc to make it convenient for absorption from the mouth andalso in the form of tablets, capsules, pills, powders etc to be absorbedfrom the stomach. Some embodiments provide a dosage form having theAmaranth extract. The dosage form is selected from the group consistingof fast melt tablet, lozenge, candy, chewing gum, beverage, tablet,capsule, pill, and powder.

Some embodiments provide a method of lowering blood pressure byadministering the extract of Amaranth. Some embodiments provide a methodof enhancing nitric oxide concentration in blood followingadministration of the Amaranth extract. Some embodiments provide, amethod of increasing physical endurance by administering an extract ofAmaranth. Some embodiments provide a method for increasing swimmingendurance by administering the Amaranth extract. Some embodimentsprovide a method for increasing running endurance by administering anAmaranth extract. Some embodiments provide a method for improving sexualperformance by administering an Amaranth extract. Some embodimentsprovide a method of enhancing nitrate level in blood by administering anextract of Amaranth. Some embodiments provide a method of enhancingnitrite level in blood by administering an extract of Amaranth. Someembodiments provide a method of enhancing nitrate level in saliva byadministering an extract of Amaranth. Some embodiments provide a methodof enhancing nitrite level in saliva by administering an extract ofAmaranth. Some embodiments provide a method of enhancing nitric oxidelevel in saliva by administering an extract of Amaranth.

In some embodiments as shown in Example 26, administering fastdissolving tablet containing Amaranth extract enriched with nitrate, ledto increased levels of NO₃, NO₂ and NO content in plasma compared toplasma levels prior to administration of tablet. In some embodiments,NO₃ level increases about 6 fold the baseline value after 1 hour. Insome embodiments, NO₂ level increases more than 6 fold. In someembodiments, NO level increases by about 6 fold.

In some embodiments as shown in Example 27, administering fastdissolving tablet containing Amaranth extract enriched with nitrate, ledto increased NO₃, NO₂ and NO levels in saliva when compared to baselineprior to administration of the tablet. In some embodiments, NO₃ levelincreases about 6.9 fold of the baseline value after 1 hour. In someembodiments, NO₂ level increases more than 9.2 fold. In someembodiments, NO level increases 7.5 fold.

In some embodiments as shown in Example 28, administering Amaranthextract enriched with nitrate mixed with water, led to increased levelsof NO₃, NO₂ and NO level in saliva when compared to levels in salivaprior to administration of extract. In some embodiments, NO₃ levelincreases 6.6 fold the baseline value after 1 hour. In some embodiments,NO₂ level increases 9.1 fold. In some embodiments, NO level increases7.3 fold.

In some embodiments, administration of Amaranth extract enhanced plasmalevel of nitrate by 2 fold to 10 fold. In some embodiment,administration of Amaranth extract enhanced plasma level of nitrite byabout 3 fold to about 15 fold. In some embodiments, administration ofAmaranth extract enhanced plasma level of nitric oxide by about 2 foldto about 10 fold.

In some embodiments, administration of Amaranth extract enhanced salivalevel of nitrate by 2 fold to 10 fold. In some embodiment,administration of Amaranth extract enhanced saliva level of nitrite byabout 3 fold to about 15 fold. In some embodiments, administration ofAmaranth extract enhanced saliva level of nitric oxide by about 2 foldto about 10 fold.

In some embodiments, administering Amaranth extract enriched withnitrate enhanced the nitrate and nitrite levels in the body. It islikely that presence of vitamin C promotes the conversion of nitrite tonitric oxide in body thereby increasing the antioxidant potential inbody. Some embodiments provide a formulation of nitrate enrichedAmaranth extract with Vitamin C, wherein administering the formulationfurther improves the generation of nitric oxide by promoting theconversion of nitrite delivered from Amaranth extract to nitric oxide inthe body. Vitamin C added to the Amaranth extract may be of synthetic ornatural origin.

The disclosure provides different extracts of Amaranth extracted byusing solvents like water, methanol, ethanol, isopropanol, n-butanol,methyl acetate, ethyl acetate, propyl acetate, n-butyl acetate andcombinations thereof

Low molecular weight alcohols that can be used in preparation of theextract include methanol, ethanol, isopropanol, n-butanol andcombinations thereof. Esters that can be used for extract preparationinclude methyl acetate, ethyl acetate, propyl acetate, n-butyl acetateand combinations thereof. Alkanes that can be used for preparation ofthe extract include pentane, hexane, heptane, isooctane, andcombinations thereof.

Various species of Amaranth from which extract can be prepared include,Amaranthus acanthochiton, Amaranthus acutilobus, Amaranthus viridis,Amaranthus albus, Amaranthus arenicola, Amaranthus australis, Amaranthusbigelovii, Amaranthus blitoides, Amaranthus blitum, Amaranthus brownie,Amaranthus californicus, Amaranthus cannabinus, Amaranthus caudatus,Amaranthus chihuahuensis, Amaranthus chlorostachys, Amaranthuscrassipes, Amaranthus crispus, Amaranthus cruentus, Amaranthus deflexus,Amaranthus dubius, Amaranthus fimbriatus, Amaranthus floridanus,Amaranthus graecizans, Amaranthus greggii, Amaranthus hybridus,Amaranthus hypochondriacus, Amaranthus leucocarpus, Amaranthus lineatus,Amaranthus lividus, Amaranthus mantegazzianus, Amaranthus minimus,Amaranthus muricatus, Amaranthus obcordatus, Amaranthus oleraceous,Amaranthus palmeri, Amaranthus paniculus, Amaranthus polygonoides,Amaranthus powellii, Amaranthus pringlei, Amaranthus pumilus, Amaranthusquitensis, Amaranthus retroflexus etc.

In some embodiments, the extract of Amaranth includes a second extract.The second extract is selected from the group consisting of amlaextract, turmeric extract, grape seed extract, green tea extract,pomegranate extract, cocoa extract, coconut root extract, rosemaryextract, mint leaf extract, star anise, sweet basil extract, cinnamonextract/clove extract, ginger extract, cumin seed extract, black pepperextract, fenugreek extract, or combinations thereof.

In some embodiments of the extract of Amaranth, the Amaranth is selectedfrom the group consisting of Amaranthus caudatus, Amaranthus cruentus,Amaranthus tricolor, Amaranthus blitum, Amaranthus viridis, Amaranthusdubis or combinations thereof.

Various methods for the preparation of the extract enriched with nitratecontent, L-arginine, flavonoids, carbohydrate, protein, saponins,alkaloids, potassium and having negligible oxalic acid or oxalatecontent prepared by the extraction of fresh leaves of Amaranth are asunder:

In some embodiments (refer FIG. 1), the fresh leaves and stem ofAmaranth is cleaned, crushed and extracted for 5 hrs using methanol at65° C. in a Soxhlet extractor and then filtered. The resultant extractis concentrated in an Agitated thin film evaporator (ATFE) at atemperature of 60° C. to form concentrated extract. Later theconcentrated extract is dried under vacuum at above 500 mm of mercury toform powder of “methanol extract” of fresh Amaranth.

In some embodiments, a method of preparing a powdered extract of freshAmaranth is provided. The method includes crushing fresh leaves and stemof Amaranth to obtain a crushed material. Then extracting the crushedmaterial with methanol to obtain a supernatant and residue. Thenfiltering the supernatant to obtain a filtrate. Then concentrating thefiltrate to obtained a concentrated filtrate. Then drying theconcentrated filtrate to obtain the powdered extract of fresh Amaranth.

According to another embodiment (refer FIG. 2), the fresh leaves andstem of Amaranth is cleaned, crushed and extracted for about 1 hr usingwater in an extractor with reflux condenser to obtain residue andsupernatant. The residue and supernatant is separated by draining outthe supernatant from the extractor bottom through the filter cloth. Theresultant supernatant is concentrated in an Agitated thin filmevaporator (ATFE) at a temperature of 85° C. to form concentratedextract. Later the concentrated extract is dried under vacuum at above500 mm of mercury to form powder of “water extract” of fresh Amaranth.

Some embodiments provide a method of preparing a powdered extract offresh Amaranth. The method includes crushing fresh leaves and stem ofAmaranth to obtain a crushed material. Then extracting the crushedmaterial with water to obtain a supernatant and a residue. Next,concentrating the supernatant to obtain a concentrated water extract.Then drying the concentrated water extract to obtain a powdered extractof fresh Amaranth.

In some embodiments (refer to FIG. 3), the fresh leaves and stem ofAmaranth is cleaned, dried and powdered. Then the powder is extractedfor 5 hrs using methanol at 65° C. in a Soxhlet extractor and thenfiltered. The resultant extract is concentrated in an Agitated thin filmevaporator (ATFE) at a temperature of 60° C. to form concentratedextract. Later the concentrated extract is dried under vacuum at above500 mm of mercury to form powder of “methanol extract” of driedAmaranth.

Some embodiments provide a method of preparing a powdered extract ofdried Amaranth. The method includes powdering dried leaves and stem ofAmaranth to obtain a powdered material. Next, the powdered material isextracted with methanol to obtain a supernatant. Then the supernatant isfiltered to obtain a filtrate. Next, concentrating the filtrate toobtain a concentrated methanol filtrate. Then drying the concentratedmethanol filtrate to obtain the powdered extract of dried Amaranth.

According to another embodiment (refer to FIG. 4), the fresh leaves andstem of amaranth is cleaned, dried and powdered. The powder is extractedfor about 1 hr using water in an extractor with reflux condenser toobtain residue and supernatant. The residue and supernatant is separatedby draining out the supernatant from the extractor bottom through thefilter cloth. The resultant supernatant is concentrated in an Agitatedthin film evaporator (ATFE) at a temperature of 85° C. to formconcentrated extract. Later the concentrated extract is dried undervacuum at above 500 mm of mercury to form powder of “water extract” ofdried Amaranth.

Some embodiments provide a method of preparing a powdered extract ofdried

Amaranth. The method includes powdering dried leaves and stem ofAmaranth to obtain a powdered material. Then, the powdered material isextracted with water to obtain a supernatant and a residue. Next, thesupernatant is concentrated to obtain a concentrated water extract. Theconcentrated water extract is dried to obtain a powdered extract ofdried Amaranth.

In another embodiment (refer to FIG. 5), the fresh leaves and stem ofamaranth is cleaned and crushed to form slurry. The slurry is a treatedwith pectinase (1%) for 2 hrs. Then the enzyme is deactivated by heatingthe slurry at 90° C. for 10 minutes. The pectinase treated amaranthslurry is extracted for about 1 hr using water in an extractor withreflux condenser to obtain residue and supernatant. The residue andsupernatant is separated by draining out the supernatant from theextractor bottom through the filter cloth. The resultant supernatant isconcentrated in an Agitated thin film evaporator (ATFE) at a temperatureof 85° C. to form concentrated extract. Later the concentrated extractis dried under vacuum at above 500 mm of mercury to form powder ofpectinase treated “water extract” of Amaranth.

In some embodiments of the method of preparing the Amaranth extract, themethod includes crushing fresh leaves and stem of Amaranth to obtain aslurry. Then treating the slurry with pectinase to obtain a pectinasetreated material. Then heating the pectinase treated material to obtaina pectinase-deactivated material. Next, extracting thepectinase-deactivated material with water to obtain a supernatant and aresidue. Then, concentrating the supernatant to obtain a concentratedsupernatant. And drying the concentrated supernatant to obtain apowdered extract of fresh Amaranth.

In yet another embodiment, (refer to FIG. 6), the fresh leaves and stemof Amaranth is cleaned and crushed to form slurry. The slurry is atreated with pectinase (1%) for 2 hrs. Then the enzyme is deactivated byheating the slurry at 90° C. for 10 minutes. The pectinase treatedAmaranth slurry is extracted for aboutl hr using water in an extractorwith reflux condenser to obtain residue and supernatant. The residue andsupernatant is separated by draining out the supernatant from theextractor bottom through the filter cloth. The resultant supernatant isconcentrated in an Agitated thin film evaporator (ATFE) at a temperatureof 85° C. to form concentrated extract. Keep the concentrated waterextract at 10° C. for 48 hr to crystallize oxalic acid or oxalates.Decant the supernatant and again keep the supernatant at 10° C. foranother 24 hr to crystallize remaining oxalic acid or oxalates. Decantand filter the supernatant and dry under vacuum at above 500 mm ofmercury to get oxalic acid or oxalate free extract of Amaranth.

In some embodiments a method of preparing an extract of Amaranth isprovided. The method includes crushing fresh leaves and stem of Amaranthto obtain a slurry, then treating the slurry with pectinase to obtain apectinase treated material, followed by heating the pectinase treatedmaterial to obtain a pectinase-deactivated material. Next thepectinase-deactivated material is extracted with water to obtain asupernatant and a residue. The supernatant is concentrated to obtain aconcentrated water extract. The concentrated water extract is cooled at10° C. for 48 hours then filtered to obtain crystals of oxalic acid oroxalate and a second supernatant. The second supernatant is cooled at10° C. for 24 hours to obtain a third supernatant and a second oxalicacid or oxalate crystals. Then the third supernatant is filtered toobtain a filtrate. Then the filtrate is concentrated to obtain aconcentrated filtrate. And the concentrated filtrate is dried to obtaina powdered extract of Amaranth. In some embodiments, the powderedextract has less than 0.1% oxalic acid.

In accordance with another embodiment (refer to FIG. 7) an Amaranthextract having enriched nitrate content is provided. A dried powder ofoxalic acid or oxalate free extract of Amaranth is obtained aftertreating with pectinase and removal of oxalic acid is further extractedwith hexane for 6 hours in a soxhlet extractor. Then filter andconcentrate the hexane extract in an Agitated thin film evaporator(ATFE). The concentrated hexane extract is dried under vacuum to getpowder of hexane extract of fresh Amaranth after treating with pectinaseand removal of oxalic acid and having enriched nitrate content.

Some embodiments provide a method of preparing an extract of Amaranthhaving enriched nitrate content. The method includes crushing freshleaves and stem of Amaranth to obtain a slurry. The slurry is treatedwith pectinase to obtain a pectinase treated material. Next, thepectinase treated material is heated to deactivate the pectinase andobtain a pectinase-deactivated material. Next, the pectinase-deactivatedmaterial is extracted with water to obtain a supernatant and a residue.Next, the supernatant is concentrated to obtain a concentrated waterextract. The concentrated water extract is cooled at 10° C. for 48 hoursand then filtered to obtain crystals of oxalic acid or oxalate and asecond supernatant. The second supernatant is cooled at 10° C. for 24hours to obtain a third supernatant and a second oxalic acid or oxalatecrystals. The third supernatant is filtered to obtain a filtrate. Thefiltrate is concentrated to obtain a concentrated filtrate. Theconcentrated filtrate is dried to obtain a powdered extract. Next thepowdered extract is treated with hexane to obtain a mixture. Then themixture is filtered to obtain a filtrate. Then the filtrate isconcentrated to obtain a concentrated hexane extract. Then theconcentrated hexane extract is dried to obtain a powdered hexaneextract.

In another embodiment (refer FIG. 8), the dried powder of oxalic acid oroxalate free extract of Amaranth obtained after treating with pectinaseand removal of oxalic acid is further extracted with chloroform by coldextraction instead of hexane. Filter and concentrate the chloroformextract in an Agitated thin film evaporator (ATFE). Then it is driedunder vacuum to get powder of chloroform extract of fresh Amaranth aftertreating with pectinase and removal of oxalic acid.

Some embodiments provide a method of preparing an extract of Amaranth.The method includes crushing fresh leaves and stem of Amaranth to obtaina slurry. Then slurry is treated with pectinase to obtain a pectinasetreated material. Then the pectinase treated material is heated toobtain a pectinase-deactivated material. Then the pectinase-deactivatedmaterial is extracted with water to obtain a supernatant and a residue.Then the supernatant is concentrated to obtain a concentrated waterextract. Then the concentrated water extract is cooled at 10 C for 48hours followed by filtering to obtain crystals of oxalic acid or oxalateand a second supernatant. Then the second supernatant is cooled at 10°C. for 24 hours to obtain a third supernatant and a second oxalic acidor oxalate crystals. Then the third supernatant is filtered to obtain afiltrate. Then the filtrate is concentrated to obtain a concentratedfiltrate. Then the concentrated filtrate is dried to obtain a powderedextract. Then the powdered extract is treated with chloroform to obtaina mixture. The mixture is filtered to obtain a filtrate. The filtrate isconcentrated to obtain a concentrated chloroform extract. Theconcentrated chloroform extract is dried to obtain a powdered chloroformextract.

According to yet another embodiment (refer to FIG. 9), the dried powderof oxalic acid or oxalate free extract of Amaranth obtained aftertreating with pectinase and removal of oxalic acid is further extractedwith ethylene dichloride (EDC) for 6 hours in a soxhlet extractorinstead of Hexane/Chloroform. Filter and concentrate the chloroformextract in an Agitated thin film evaporator (ATFE). Then it is driedunder vacuum to get powder of chloroform extract of fresh Amaranth aftertreating with pectinase and removal of oxalic acid.

Some embodiments provide a method of preparing an extract of Amaranth.The method includes crushing fresh leaves and stem of Amaranth to obtaina slurry. The slurry is treated with pectinase to obtain a pectinasetreated material. The pectinase treated material is heated to obtain apectinase-deactivated material. The pectinase-deactivated material isextracted with water to obtain a supernatant and a residue. Thesupernatant is concentrated to obtain a concentrated water extract. Theconcentrated water extract is cooled at 10° C. for 48 hours followed byfiltering to obtain crystals of oxalic acid or oxalate and a secondsupernatant. The second supernatant is cooled at 10° C. for 24 hours toobtain a third supernatant and a second set of crystals of oxalic acidor oxalate. The third supernatant is filtered to obtain a filtrate. Thefiltrate is concentrated to obtain a concentrated filtrate. Theconcentrated filtrate is dried to obtain a powdered extract. Thepowdered extract is dried with ethylene dichloride to obtain a mixture.The mixture is filtered to obtain a second filtrate. The second filtrateis concentrated to obtain a concentrated ethylene dichloride extract.The concentrated ethylene dichloride extract is dried to obtain apowdered ethylene dichloride extract.

In another embodiment (refer to FIG. 11), the dried powder of oxalicacid or oxalate free extract of Amaranth obtained after treating withpectinase and removal of oxalic acid is further extracted with hexanefor 6 hours in a soxhlet extractor. Then filter and concentrate thehexane extract in an Agitated thin film evaporator (ATFE). Theconcentrated hexane extract is dried under vacuum to get powder ofhexane extract. It is dissolved in methanol and Amla extract alreadyprepared is added and stirred for 1 hr at 100 rpm using mechanicalstirrer. Evaporate and dry under vacuum to form powder. Thus a hexaneextract of fresh Amaranth after treating with pectinase and removal ofoxalic acid in combination with Amla is obtained.

The Amla extract (refer to FIG. 10) used here can be prepared accordingto the following method.

Pulp the fruits of amla and extract the pulp using methanol at 65° C. ina Soxhlet extractor for 5 hrs. The resultant methanol extract isfiltered and concentrated in an Agitated thin film evaporator (ATFE) ata temperature of 60° C. to form concentrated extract. Dry theconcentrated extract under vacuum at above 500 mm of mercury to formpowder of methanol extract of Amla.

Some embodiments provide a method of preparing an extract of Amaranth.The method includes crushing fresh leaves and stem of Amaranth to obtaina slurry. Then the slurry is treated with pectinase to obtain apectinase treated material. The pectinase treated material is heated toobtain a pectinase-deactivated material. The pectinase-deactivatedmaterial is extracted with water to obtain a supernatant and a residue.The supernatant is concentrated to obtain a concentrated water extract.The concentrated water extract is cooled at 10° C. for 48 hours followedby filtering to obtain crystals of oxalic acid or oxalate and a secondsupernatant. The second supernatant is cooled at 10° C. for 24 hours toobtain a third supernatant and a second oxalic acid or oxalate crystals.The third supernatant is filtered to obtain a filtrate. The filtrate isconcentrated to obtain a concentrated filtrate. The concentratedfiltrate is dried to obtain a powdered extract. The powdered extract istreated with hexane to obtain a mixture. The mixture is filtered toobtain a second filtrate. The second filtrate is concentrated to obtaina concentrated hexane extract. The concentrated hexane extract is driedto obtain a powdered hexane extract. The powdered hexane extract isdissolved in methanol to obtain a solution of hexane extract ofAmaranth. A powder of methanol extract of Amla is added to the solutionof hexane extract of Amaranth to obtain a second mixture. The secondmixture is evaporated to obtain an evaporate. The evaporate is dried toobtain a powder of Amaranth and Amla extract. The powdered methanolextract of Amla extract is prepared by a process including pulping freshAmla (Emblica officinalis) fruit to obtain a pulp. The pulp is extractedwith methanol to obtain a filtrate. The filtrate is concentrated toobtain a concentrated methanol extract of Amla. The concentratedmethanol extract of Amla is dried to obtain the powdered methanolextract of Amla.

In some embodiments, amla extract/turmeric extract/grape seedextract/green tea extract/pomegranate extract/cocoa extract/coconut rootextract/rosemary extract/mint leaf extract/star anise/sweet basilextract/cinnamon extract/clove extract/ginger extract/cumin seedextract/black pepper extract/fenugreek extract are added to thepectinase treated fresh leaves of Amaranth having low total oxalic acidor oxalate content to fortify the Amaranth extract. The combinationextracts can be administered to a mammal for enhancing the amount ofnitric oxide in blood.

In some embodiments (refer to FIG. 13), the fresh leaves of amaranth iscleaned and crushed in the screw expeller. Initial Juice obtained on thetray of the screw expeller is collected in the feed tank under roomtemperature of 27° C. Initial juice is centrifuged to obtain filtrateand residue is removed. Filtrate is clarified in a rotating disc typeclarifier (RPM—18000) to obtain final filtrate and residue. Residue isremoved and final filtrate is collected (clarified liquid juice ofleaves of Amaranth).

Details of some of the trials/experiments carried out and findings areexplained below by way of examples.

EXAMPLE 1

Preparation of Amaranth Fresh Leaves Extract with Methanol.

Fresh Amaranth were collected (100 Kg). Leaves and stem of freshAmaranth were cleaned and crushed. Crushed materials were filled in theSoxhlet extractor and extracted with methanol (300 L). The extractionwas carried out for 5 hrs at a temperature of about 65° C. After thecompletion of extraction, the supernatant was filtered and concentratedin an Agitated thin film evaporator (ATFE) at a temperature of 60° C. toform concentrated methanol extract. Concentrated methanol extract wasdried under vacuum at above 500 mm of mercury to get powder of methanolextract of fresh Amaranth (Yield 2 Kg). See also extract preparation inFIG. 1.

The nitrate content in methanol extract of fresh Amaranth by ionchromatography was found to be 1.5%.

EXAMPLE 2

Preparation of Amaranth Fresh Leaves Extract with Water.

Fresh Amaranth were collected (100 Kg). Leaves and stem of freshAmaranth were cleaned and crushed. Water in an amount ten times thequantity of crushed material of Amaranth was added to form a mixture.The extraction was performed using an extractor with a reflux condenser.The bottom of the extractor was fitted with a polypropylene (100microns) filter cloth. The mixture was refluxed for one hour to obtain afirst residue and supernatant. The residue and supernatants wereseparated by draining out the supernatant from the extractor bottomthrough the polypropylene filter cloth using a centrifugal pump. Afterthe first extraction, the first residue was further extracted with tentimes the quantity of water to get second residue and supernatant. Thesecond residue was further extracted with ten times the quantity ofwater to get third residue and supernatant. All the supernatants werepooled and concentrated in an Agitated thin film evaporator (ATFE) at atemperature of 85° C. to form concentrated water extract. Concentratedwater extract was dried under vacuum at above 500 mm of mercury to getpowder of water extract of fresh Amaranth. (Yield 3%). See also extractpreparation in FIG. 2.

The nitrate content in water extract of fresh Amaranth by ionchromatography was found to be 1.6%.

EXAMPLE 3

Preparation of Amaranth Dried Leaves Extract with Methanol.

Fresh Amaranth were collected (100 Kg). Leaves and stem of freshAmaranth were cleaned and dried. Dried leaves and stem were powdered.Powdered Amaranth were filled in the Soxhlet apparatus and extractedwith methanol (500 L). The extraction was carried out for 5 hrs at atemperature of about 65° C. After the completion of extraction, thesupernatant was filtered and concentrated in an Agitated thin filmevaporator (ATFE) at a temperature of 60° C. to form concentratedmethanol extract. Concentrated methanol extract was dried under vacuumat above 500 mm of mercury to get powder of methanol extract of driedamaranth. When starting with 100 kg of fresh amaranth, a yield of 24 Kgof powder of methanol extract of dried amaranth was obtained. See alsoextract preparation in FIG. 3.

The nitrate content in methanol extract of dried Amaranth by ionchromatography was found to be 1.65%.

EXAMPLE 4

Preparation of Amaranth Dried Leaves Extract ith Water.

Fresh Amaranth were collected (100 Kg). Leaves and stem of freshAmaranth were cleaned and dried. Dried Amaranth was powdered. Water inan amount ten times the quantity of powder of dried Amaranth was addedto the powder of Amaranth to form a mixture. The extraction wasperformed using an extractor with a reflux condenser. The bottom of theextractor was fitted with a polypropylene (100 microns) filter cloth.The mixture was refluxed for one hour to obtain a first residue andsupernatant. The residue and supernatants were separated by draining outthe supernatant from the extractor bottom through the polypropylenefilter cloth using a centrifugal pump. After the first extraction, thefirst residue was further extracted with ten times the quantity of waterto get second residue and supernatant. The second residue was furtherextracted with ten times the quantity of water to get third residue andsupernatant. All the supernatants were pooled and concentrated in anAgitated thin film evaporator (ATFE) at a temperature of 85° C. to formconcentrated water extract. Concentrated water extract was dried undervacuum at above 500 mm of mercury to get powder of water extract ofdried Amaranth. (Yield 25%). See also extract preparation in FIG. 4.

The nitrate content in water extract of dried Amaranth by ionchromatography was found to be 1.7%.

EXAMPLE 5

Enhanced Yield of Amaranth Extract by Treatment with Pectinase.

Fresh Amaranth were collected (100 Kg). Leaves and stem of freshAmaranth were cleaned and crushed to make slurry. The slurry was treatedwith pectinase (1%) for 2 Hrs. The enzyme was deactivated by heating theslurry at 90° C. for 10 minutes. After the enzyme treatment, thepectinase treated fresh leaves were extracted with water. The extractionwas performed with water using an extractor with a reflux condenser. Thebottom of the extractor was fitted with a polypropylene (100 microns)filter cloth. The mixture was refluxed for one hour to obtain a firstresidue and supernatant. The residue and supernatants were separated bydraining out the supernatant from the extractor bottom through thepolypropylene filter cloth using a centrifugal pump. After the firstextraction, the first residue was further extracted with ten times thequantity of water to get second residue and supernatant. The secondresidue was further extracted with ten times the quantity of water toget third residue and supernatant. All the supernatants were pooled andconcentrated in an Agitated thin film evaporator (ATFE) at a temperatureof 85° C. to form concentrated water extract. Concentrated water extractwas dried under vacuum at above 500 mm of mercury to get powder of waterextract of fresh Amaranth (Yield 3.5 Kg). See also extract preparationin FIG. 5.

Nitrate content of the extract was found to be 2% as determined by ionchromatography.

EXAMPLE 6

Oxalic Acid or Oxalates Free Amaranth Extract.

Fresh Amaranth were collected (100 Kg). Leaves and stem of freshAmaranth were cleaned and crushed to make slurry. The slurry was treatedwith pectinase (1%) for 2 Hrs. The enzyme was deactivated by heating theslurry at 90° C. for 10 minutes. After the enzyme treatment, thepectinase treated fresh leaves were extracted with water. The extractionwas performed with water using an extractor with a reflux condenser. Thebottom of the extractor was fitted with a polypropylene (100 microns)filter cloth. The mixture was refluxed for one hour to obtain a firstresidue and supernatant. The residue and supernatants were separated bydraining out the supernatant from the extractor bottom through thepolypropylene filter cloth using a centrifugal pump. After the firstextraction, the first residue was further extracted with ten times thequantity of water to get second residue and supernatant. The secondresidue was further extracted with ten times the quantity of water toget third residue and supernatant. All the supernatants were pooled andconcentrated in an Agitated thin film evaporator (ATFE) at a temperatureof 85° C. to form concentrated water extract.

Concentrated water extract was kept at 10° C. for 48 hr to crystallizeoxalic acid or oxalates. The supernatant was decanted off and again keptat 10° C. for 24 hr to crystallize remaining oxalic acid or oxalates.The supernatant from this solution was decanted, filtered and evaporatedto dryness under vacuum to get oxalic acid or oxalate free Amaranthextract (2.2 Kg). See also extract preparation in FIG. 6

Nitrate content of the extract was found to be 5% as determined by ionchromatography.

EXAMPLE 7

Enrichment of Nitrates in Amaranth Extract by Extraction with Hexane.

Fresh Amaranth were collected (100 Kg). Leaves and stem of freshAmaranth were cleaned and crushed to make slurry. The slurry was treatedwith pectinase (1%) for 2 Hrs. The enzyme was deactivated by heating theslurry at 90° C. for 10 minutes. The extraction was performed with waterusing an extractor with a reflux condenser. The bottom of the extractorwas fitted with a polypropylene (100 microns) filter cloth. The mixturewas refluxed for one hour to obtain a first residue and supernatant. Theresidue and supernatants were separated by draining out the supernatantfrom the extractor bottom through the polypropylene filter cloth using acentrifugal pump. After the first extraction, the first residue wasfurther extracted with ten times the quantity of water to get secondresidue and supernatant. The second residue was further extracted withten times the quantity of water to get third residue and supernatant.All the supernatants were pooled and concentrated in an Agitated thinfilm evaporator (ATFE) at a temperature of 85° C. to form concentratedwater extract.

Concentrated water extract was kept at 10° C. for 48 hr to crystallizeoxalic acid or oxalates. The supernatant was decanted off and again keptat 10° C. for 24 hr to crystallize remaining oxalic acid or oxalates.The supernatant from this solution was decanted, filtered and evaporatedto dryness under vacuum to get oxalic acid or oxalate free Amaranthextract (2.2 Kg).

This oxalic acid or oxalate free Amaranth extract was further extractedwith hexane for 6 hours in a soxhlet extractor. The extract was filteredand evaporated to dryness under vacuum to get nitrate enriched Amaranthextract (1.6 Kg). Nitrate content of the extract was found to be 18% asdetermined by ion chromatography. See also extract preparation in FIG.7.

EXAMPLE 8

Enrichment of Nitrates in Amaranth Extract by Extraction withChloroform.

Fresh Amaranth were collected (100 Kg). Leaves and stem of freshAmaranth were cleaned and crushed to make slurry. The slurry was treatedwith pectinase (1%) for 2 Hrs. The enzyme was deactivated by heating theslurry at 90° C. for 10 minutes. The extraction was performed with waterusing an extractor with a reflux condenser. The bottom of the extractorwas fitted with a polypropylene (100 microns) filter cloth. The mixturewas refluxed for one hour to obtain a first residue and supernatant. Theresidue and supernatants were separated by draining out the supernatantfrom the extractor bottom through the polypropylene filter cloth using acentrifugal pump. After the first extraction, the first residue wasfurther extracted with ten times the quantity of water to get secondresidue and supernatant. The second residue was further extracted withten times the quantity of water to get third residue and supernatant.All the supernatants were pooled and concentrated in an Agitated thinfilm evaporator (ATFE) at a temperature of 85° C. to form concentratedwater extract.

Concentrated water extract was kept at 10° C. for 48 hr to crystallizeoxalic acid or oxalates. The supernatant was decanted off and again keptat 10° C. for 24 hr to crystallize remaining oxalic acid or oxalates.The supernatant from this solution was decanted, filtered and evaporatedto dryness under vacuum to get oxalic acid or oxalate free Amaranthextract (2.2 Kg).

This oxalic acid or oxalate free Amaranth extract was further extractedwith chloroform by cold extraction. The extract was filtered andevaporated to dryness under vacuum to get nitrate enriched Amaranthextract (1.6 Kg). See also extract preparation in FIG. 8.

Nitrate content of the extract was found to be 16% as determined by ionchromatography.

EXAMPLE 9

Enrichment of Nitrates in Amaranth Extract by Extraction with EthyleneDichloride (EDC).

Fresh Amaranth were collected (100 Kg). Leaves and stem of freshAmaranth were cleaned and crushed to make slurry. The slurry was treatedwith pectinase (1%) for 2 Hrs. The enzyme was deactivated by heating theslurry at 90° C. for 10 minutes. The extraction was performed with waterusing an extractor with a reflux condenser. The bottom of the extractorwas fitted with a polypropylene (100 microns) filter cloth. The mixturewas refluxed for one hour to obtain a first residue and supernatant. Theresidue and supernatants were separated by draining out the supernatantfrom the extractor bottom through the polypropylene filter cloth using acentrifugal pump. After the first extraction, the first residue wasfurther extracted with ten times the quantity of water to get secondresidue and supernatant. The second residue was further extracted withten times the quantity of water to get third residue and supernatant.All the supernatants were pooled and concentrated in an Agitated thinfilm evaporator (ATFE) at a temperature of 85° C. to form concentratedwater extract.

Concentrated water extract was kept at 10° C. for 48 hr to crystallizeoxalic acid or oxalates. The supernatant was decanted off and again keptat 10° C. for 24 hr to crystallize remaining oxalic acid or oxalates.The supernatant from this solution was decanted, filtered and evaporatedto dryness under vacuum to get oxalic acid or oxalate free Amaranthextract (2.2 Kg).

This oxalic acid or oxalate free Amaranth extract was further extractedwith EDC for 8 hours in a soxhlet extractor. The extract was filteredand evaporated to dryness under vacuum to get nitrate enriched Amaranthextract (1.6 Kg). See also extract preparation in FIG. 9.

Nitrate content of the extract was found to be 17% as determined by ionchromatography.

EXAMPLE 10

Preparation of Amla Extract.

Fresh Amla fruits weighing 10 Kg were pulped and the pulp was extractedwith methanol at 65° C. for 8 hours in a soxhlet apparatus. After thecompletion of extraction, the supernatant was filtered and concentratedat 60° C. to form concentrated methanol extract of Amla. Concentratedmethanol extract was dried under vacuum at above 500 mm of mercury toget methanol extract of fresh Amla fruits (Yield: 500 gm). See alsoextract preparation in FIG. 10.

EXAMPLE 11

Amaranth eExtract Enriched with Amla Extract.

1.5 Kg of fresh Amaranth leaves extract (from Example 7) was taken in abeaker and dissolved in methanol (5 L). 150 gm of Amla extract (fromExample 10) was added to this solution and stirred for 1 hr at 100 rpmusing mechanical stirrer. The obtained mixture was evaporated to drynessunder vacuum to get combined extract. See also extract preparation inFIG. 11

Nitrate content of this combined extract was 17%.

EXAMPLE 12

Method of Preparation of Amaranth Leaf Powder.

Fresh Amaranth were collected (100 Kg). Leaves of fresh Amaranth werecleaned and dried. Dried leaves were powdered to get Amaranth leafpowder. See also extract preparation in FIG. 12

EXAMPLE 13

Method of Preparation of Juice from Amaranth Leaves.

Fresh Amaranth leaves were collected (2 Kg). Leaves of fresh Amaranthwere cleaned. The cleaned leaves were crushed in the screw expeller. Theinitial juice obtained on the tray of the screw expeller was collectedin the feed tank under room temperature of 27° C. The initial juice wascentrifuged to obtain filtrate which was collected and the residue whichwas removed. The collected filtrate was clarified in a rotating disctype clarifier (RPM—18000) to obtain final filtrate and some residue.After removing the residue, the final filtrate (clarified liquid juiceof leaves of Amaranth) was collected with a yield 750 ml juice from 2 KgAmaranth leaves. See also extract preparation in FIG. 13.

EXAMPLE 14

Preparation of Methanol Extract of Amaranth Fresh Leaves Using DifferentVarieties of Amaranth.

Fresh leaves and stem of Amaranthus tricolor were collected (100 Kg).Leaves and stem of fresh Amaranthus tricolor were cleaned and crushed.Crushed materials were filled in the Soxhlet extractor and extractedwith methanol (300 L). The extraction was carried out for 5 hrs at atemperature of about 65° C. After the completion of extraction, thesupernatant was filtered and concentrated in an Agitated thin filmevaporator (ATFE) at a temperature of 60° C. to form concentratedmethanol extract. Concentrated methanol extract was dried under vacuumat above 500 mm of mercury to get powder of methanol extract of freshAmaranthus tricolor (Product 1) (Yield 2.1 Kg).

The nitrate content in methanol extract of fresh Amaranthus tricolour byHPLC was found to be 1.48%.

Methanol extract of different species of Amaranth were prepared followedby the same procedure as mentioned above.

Different species of Amaranthus that were used for the extraction areAmaranthus caudatus, Amaranthus cruentus, Amaranthus blitum andAmaranthus viridis. After methanol extraction of different speciesyields

-   -   1. Powder of methanol extract of fresh Amaranthus caudatus        (Product II) (Yield 1.95 Kg).        -   The nitrate content in methanol extract of fresh Amaranthus            caudatus by HPLC was found to be 1.55%.    -   2. Powder of methanol extract of fresh Amaranthus cruentus        (Product III) (Yield 2 Kg).        -   The nitrate content in methanol extract of fresh Amaranthus            cruentus by HPLC was found to be 1.53%.    -   3. Powder of methanol extract of fresh Amaranthus blitum        (Product IV) (Yield 2.05 Kg).        -   The nitrate content in methanol extract of fresh Amaranthus            blitum by HPLC was found to be 1.5%.    -   4. Powder of methanol extract of fresh Amaranthus viridis        (Product V) (Yield 2.15 Kg).        -   The nitrate content in methanol extract of fresh Amaranthus            viridus by HPLC was found to be 1.49%.

EXAMPLE 15

Enrichment of Nitrates in Amaranth (Different Varieties) Extract byExtraction with Hexane.

Fresh leaves and stem of Amaranthus tricolor were collected (100 Kg).Leaves and stem of fresh Amaranthus tricolor were cleaned and crushed tomake slurry. The slurry was treated with pectinase (1%) for 2 Hrs. Theenzyme was deactivated by heating the slurry at 90° C. for 10 minutes.The extraction was performed with water using an extractor with a refluxcondenser. The bottom of the extractor was fitted with a polypropylene(100 microns) filter cloth. The mixture was refluxed for one hour toobtain a first residue and supernatant. The residue and supernatantswere separated by draining out the supernatant from the extractor bottomthrough the polypropylene filter cloth using a centrifugal pump. Afterthe first extraction, the first residue was further extracted with tentimes the quantity of water to get second residue and supernatant. Thesecond residue was further extracted with ten times the quantity ofwater to get third residue and supernatant. All the supernatants werepooled and concentrated in an Agitated thin film evaporator (ATFE) at atemperature of 85° C. to form concentrated water extract.

Concentrated water extract was kept at 10° C. for 48 hr to crystallizeoxalic acid or oxalates. The supernatant was decanted off and again keptat 10° C. for 24 hr to crystallize remaining oxalic acid or oxalates.The supernatant from this solution was decanted, filtered and evaporatedto dryness under vacuum to get oxalic acid or oxalate free extract ofAmaranthus tricolor (2.22 Kg).

This oxalic acid or oxalate free extract of Amaranthus tricolor wasfurther extracted with hexane for 6 hours in a soxhlet extractor. Theextract was filtered and evaporated to dryness under vacuum to getnitrate enriched extract of Amaranthus tricolor (Product I) (1.63 Kg).

Nitrate content of the extract was found to be 17.7% as determined byHPLC.

Nitrate enriched extract of different species of Amaranthus wereprepared followed by the same procedure as mentioned above.

Different species of Amaranthus that were used for the extraction areAmaranthus caudatus, Amaranthus cruentus, Amaranthus blitum andAmaranthus viridis. After extraction of different species yields

-   -   1. Powder of nitrate enriched extract of fresh Amaranthus        caudatus (Product II) (Yield 1.62 Kg)    -   The nitrate content in nitrate enriched extract of fresh        Amaranthus caudatus by HPLC was found to be 17.9%.    -   2. Powder of nitrate enriched extract of fresh Amaranthus        cruentus (Product III) (Yield 1.6 Kg)    -   The nitrate content in nitrate enriched extract of fresh        Amaranthus cruentus by HPLC was found to be 18%.    -   3. Powder of nitrate enriched extract of fresh Amaranthus blitum        (Product IV) (Yield 1.58 Kg)    -   The nitrate content in nitrate enriched extract of fresh        Amaranthus blitum by HPLC was found to be 18.3%.    -   4. Powder of nitrate enriched extract of fresh Amaranthus        viridis (Product V) (Yield 1.61 Kg)    -   The nitrate content in nitrate enriched extract of fresh        Amaranthus viridus by HPLC was found to be 18.1%.

EXAMPLE 16

Determination of Nitrate and Nitrite Content in Amaranth by IonChromatography.

A 50 microliter aliquot of sample was pipette into a 300 microliterglass tube (Sci-Vi, Chromacol, Welwyn, UK). Acetonitrile (50 microliter)was added, and the tube with an autosampler limited-volume insert springwas put in a 4-ml WISP vial, capped, mixed and centrifuged at 2000 g for2 min. The supernatant was injected for analysis.

882 Compact IC plus chromatograph (Metrohm, USA) fitted with a WISPautosampler (Waters, Watford, UK). The column was a Metrosep A Supp5-150/4.0 ion-exchange column, 150 mm×4 mm (Metrohm), and the eluent was1.7 mM Sodium bicarbonate (NaHCO₃), 1.8 mM Sodium carbonate (Na₂CO₃),pumped at 1.5 ml/min. Detection was by conductivity (DX100) andabsorbance at 214 nm using a 441 detector fitted with a zinc lamp(Waters). Data was acquired and processed using an 840 data system(Waters).

EXAMPLE 17

Determination of Nitrate and Nitrite by HPLC Method.

Standard for nitrate (NO₃) was prepared by weighing 81.53 mg potassiumnitrate (99% pure) in to 100 ml standard flask and made up to mark withwater. 1 ml from the 100 ml solution was pipetted out into 50 mlstandard flask and made up to mark with water.

Standard for nitrite (NO₂) was prepared by weighing 92.49 mg potassiumnitrite (96% pure) in to 100 ml standard flask and made up to mark withwater. 1 ml from the 100 ml solution was pipetted out into 50 mlstandard flask and made up to mark with water. Again 1 ml from the 50 mlsolution was pipetted out into 50 ml standard flask and again made up tomark with water.

Added 1 ml standard of nitrite and 1 ml mobile phase A (Weighed 1.3 g ofthe Tetra Butyl ammonium Hydroxide Solution (10% aqu) in to 1000 mlstandard flask. Made up to mark with Water. Then PH adjusted to 2.50with Con: H₂SO₄), 200μ Griess A (Weighed 50.00 mg sulphanilic acid and1.5 ml acetic acid and 3 ml purified water was added and dissolved) andafter 2 min added 200μ Griess B (Weighed 4.00 mg 1-naphthylamine and 4.0ml acetic acid was added and dissolved) in to a plastic tube. Filteredthrough 0.45 μm membrane filter before injection.

Sample (Amaranth extract) was prepared by weighing accurately 20-25 mgof the extract powder to 25 ml std flask and made up the withdemineralized water. Filtered through 0.45 Micron membrane filter anddivided into 2 portions. One portion was used for Nitrate and otherportion was used for Nitrite determinations.

NO₃ was analyzed by HPLC on a BETASIL C-18 column (250×4.6 mm) usingTetra butyl ammonium hydroxide in De-ionized water, acetonitrile andmethanol as mobile phase and Photo diode array detection at 222 nm. NO₂was analysed at 520 nm using the same conditions.

${{Nitrite}\mspace{14mu}\%} = \frac{\begin{matrix}{{Area}\mspace{14mu}{of}\mspace{14mu}{Nitrite}\mspace{14mu}{peak}\mspace{14mu}{in}\mspace{14mu}{sample} \times {Amount}\mspace{14mu}{of}\mspace{14mu}{Std} \times} \\{{Purity}\mspace{14mu}{of}\mspace{14mu}{Std}}\end{matrix}}{{{Area}\mspace{14mu}{of}\mspace{14mu}{Nitrite}\mspace{14mu}{peak}\mspace{14mu}{in}\mspace{14mu}{Standard}\; \times {Weight}\mspace{14mu}{of}\mspace{14mu}{the}\mspace{14mu}{Sample}}\mspace{14mu}}$${{Nitrate}\mspace{14mu}\%} = \frac{\begin{matrix}{{Area}\mspace{14mu}{of}\mspace{14mu}{Nitrate}\mspace{14mu}{peak}\mspace{14mu}{in}\mspace{14mu}{sample} \times {Amount}\mspace{14mu}{of}\mspace{14mu}{Std} \times} \\{{Purity}\mspace{14mu}{of}\mspace{14mu}{Std}}\end{matrix}}{{Area}\mspace{14mu}{of}\mspace{14mu}{Nitrate}\mspace{14mu}{peak}\mspace{14mu}{in}\mspace{14mu}{Standard} \times {Weight}\mspace{14mu}{of}\mspace{14mu}{the}\mspace{14mu}{Sample}}$

EXAMPLE 18

Determination of Nitric Oxide (NO).

Total NO content in plasma can be interpreted as the sum of totalnitrate (NO₃) and nitrite (NO₂) in plasma.

(Journal of Medical sciences (2010):3 (3): 153-159, Plasma Endothelin—1,Homocysteine, and Oxide Levels in a Multiethnic Hypertensive Cochot fromthe United Arab Emirates.)

NO levels was recorded as the sum of the two major metabolites of NO,nitrite and nitrate.

EXAMPLE 19

Determination of Total Oxalic Acid Content.

Oxalic acid was estimated by high performance liquid chromatography(HPLC-DAD) on a C18 column (250×4.6 mm, Gemini 5 μm, USA.). The mobilephase was 5 mM H2SO4 and used under isocrating condition with an eluentflow rate of 1 ml/min. Oxalic acid was detected at 210 nm.

Standard was prepared by weighing 5 mg of standard oxalic acid (95%purity) and was made up to 50 ml with 6 mM H2SO4. Sample was prepared byweighing 50 mg of the dry extract of Amaranth and was made up to 50 mlwith 6 mM H2SO4. Both the sample and standard were filtered separatelythrough a 0.2 μm membrane filter before injection into the HPLC column.The injection volume was 20 μl. Oxalic acid was detected at 210 nm. Bycomparing the area of standard and sample, the percentage of oxalic acidpresent in the sample was quantified.

${{Oxalic}\mspace{14mu}{acid}\mspace{14mu}\%} = \frac{{{Area}\mspace{14mu}{of}\mspace{14mu}{sample} \times {amount}\mspace{14mu}{of}\mspace{14mu}{standard} \times {purity}\mspace{14mu}{of}\mspace{11mu}{standard}}\;}{{Amount}\mspace{14mu}{of}\mspace{14mu}{sample} \times {Area}\mspace{14mu}{of}\mspace{14mu}{standard}}$

EXAMPLE 20

Determination of Potassium Content.

Potassium was determined by Atomic absorption spectroscopy (AAS). A 100ppm solution of potassium Standard (KCl) was prepared. Seven differentstandards of potassium in different concentrations were prepared bysimple dilution of the above 100 ppm solution. The sample solution andstandard were measured in the AAS machine at 766.5 nm to create acalibration curve for determination of potassium content in the samplesolutions.

EXAMPLE 21

Preparation of Fast Dissolving Tablets.

Fast dissolving tablets were made by mixing Fast-Melt (119.2 gm) withamaranth nitrate enriched (18% nitrate) extract (80 gm) (as per Example7). Magnesium stearate (0.8 gm) was added to the mixture before tabletpunching. Tablets weighing 200 mg were punched on automatic tablet punchmachine using 8 mm die to get 5 Kg hardness. The final composition ofeach tablet was 119.2 mg F-Melt, 80 mg extract (approx. 14.4 mg nitrate)and 0.8 mg of magnesium stearate. Disintegration time of tablets wasapproximately 30 seconds.

EXAMPLE 22

Preparation of Fast Dissolving Tablets.

Fast dissolving tablets were made by mixing Fast-Melt (745 gm) withamaranth nitrate enriched (18% nitrate) extract (500 gm) prepared as perexample 7. Magnesium stearate (5 gm) was added to the mixture beforetablet punching. Tablets weighing 1250 mg were punched on automatictablet punch machine using 12 mm die to get 5 Kg hardness. The finalcomposition of each tablet was 745 mg F-Melt, 500 mg extract (approx. 90mg nitrate) and 5 mg of magnesium stearate. Disintegration time oftablets was approximately 30 seconds.

Similarly fast melt tablets were made by substitute the above extractwith

-   -   1. Methanol extract of fresh leaves of Amaranth (1.5% nitrate)        (Final composition of Fast melt tablet contain 750 mg F-Melt,        500 mg extract (approx 7.5 mg nitrate) and 5 mg of magnesium        stearate)    -   2. Water extract of fresh leaves of Amaranth (1.6% nitrate)        (Final composition of Fast melt tablet contain 750 mg F-Melt,        500 mg extract (approx 8 mg nitrate) and 5 mg of magnesium        stearate)    -   3. Methanol extract of dried leaves of Amaranth (1.65% nitrate)        (Final composition of Fast melt tablet contain 750 mg F-Melt,        500 mg extract (approx 8.25 mg nitrate) and 5 mg of magnesium        stearate)    -   4. Water extract of dried leaves of Amaranth (1.7% nitrate)        (Final composition of Fast melt tablet contain 750 mg F-Melt,        500 mg extract (approx 8.5 mg nitrate) and 5 mg of magnesium        stearate)    -   5. Pectinase treated water extract of fresh leaves of Amaranth        (2% nitrate) (Final composition of Fast melt tablet contain 750        mg F-Melt, 500 mg extract (approx 10 mg nitrate) and 5 mg of        magnesium stearate)    -   6. Extract of Amaranthus blitum with enriched nitrate (18.3%        nitrate) (Final composition of Fast melt tablet contain 750 mg        F-Melt, 500 mg extract (approx 91.5 mg nitrate) and 5 mg of        magnesium stearate)    -   7. Extract of Amaranthus viridus with enriched nitrate (18.1%        nitrate) (Final composition of Fast melt tablet contain 750 mg        F-Melt, 500 mg extract (approx 90.5 mg nitrate) and 5 mg of        magnesium stearate)

EXAMPLE 23

Preparation of Chewing Gum.

Chewing gums were prepared by adding amaranth nitrate enriched extract(80 mg for one chewing gum) to the base material (920 mg for one chewinggum). The base material consists of polyvinyl acetate in an amount byweight of about 38%, natural rubber in an amount by weight of about 10%,partially hydrogenated soybean oil in an amount by weight of about 11%,polyisobutylene in an amount by weight of about 12%, dicalcium phosphatein an amount by weight of about 13.92%, triacetin in an amount by weightof about 3%, mono-di-glycerides in an amount by weight of about 7%,liquid glucose in an amount by weight of about 5%, and BHT (butylatedhydroxyl toluene) in an amount by weight of about 0.08%.

The chewing gum components (except liquid glucose and BHT) were mixed ina mixer with strong horizontally placed Z-blades, which processes thecomponents into a homogeneous gum mass. The mixer was heated to atemperature of approx. 50-60° C. The mixing process starts with mixingand melting the gum base for 3 minutes.

The liquid glucose was then added and mixed for 4 minutes. Subsequent,the amaranth nitrate enriched extract were added and the mixture wasmixed for further 6 minutes. The BHT was then added and mixed forfurther 5 minutes.

When the mixing was completed, the gum mass was taken out onto trays androlled out to a 1-2 cm thick sheet. The chewing gum mass was subsequentcooled for approx. 15-20 minutes until the right texture of the gum masswas achieved.

The formed and cooled chewing gum cores were then coated and polishedwith a coating suspension in a round stainless steel coating kettle thatrotate during the coating and polishing process. The coating suspensionconsists of Xylitol (58.2%), Mannitol (10.6%), Gelatin (1.2%), Titaniumdioxide (0.9%), Acesulfame K (0.1%) and water (29%).

The polishing was done in rotating kettles in which carnauba wax (0.1%)was added to the coated chewing gums in one portion. Polishing was doneuntil a shinning surface is achieved, typically for 10-20 minutes. Eachchewing gum contained 80 mg of extract (approx. 14.4 mg nitrate).

EXAMPLE 24

Nitric Oxide Level in Rats.

18 albino rats (Sprague-Dawley (SD) strain) weighing 200-250 gm weretaken for the study. The animals were acclimatized to the animal housecondition (24±2° C. temperature, 65% relative humidity, 12 hr light/darkcycle) for one week. The animals were divided into three groupscomprising of six animals in each group. The animals were fastedovernight and blood was collected to determine the baseline nitric oxidelevel. The animals were treated as follows:

-   Group 1: Vehicle control-   Group 2: Amaranth dried leaves powder—1 gm/kg-   Group 3: Amaranth nitrate enriched extract (from example 7)—100    mg/kg

After 30 minutes of feeding, blood was collected from all the animals inEDTA (ethylenediaminetetraaceticacid) coated tubes and plasma wasseparated immediately by centrifuging the tubes at 4000 rpm (rotationsper minute) for 10 minutes. The plasma was stored below −80° C. untilanalysis.

The nitric oxide (NO) content of the plasma was reported as μmol/L.

Baseline Nitric Nitric oxide oxide level level after 30 min S. No.Groups (μmol/L) (μmol/L) 1. Vehicle control 18.2 18.4 2. Amaranth driedleaves 17.9 18.2 powder 3. Amaranth nitrate enriched 18.6 42.3 extract(from example 7)

EXAMPLE 25

Nitric Oxide Level in Mice.

18 swiss albino mice weighing 25-30 gm were taken for the study. Theanimals were acclimatized to the animal house condition (24±2° C.temperature, 65% relative humidity, 12 hr light/dark cycle) for oneweek. The animals were divided into three groups comprising of sixanimals in each group. The animals were fasted overnight and blood wascollected to determine the baseline nitric oxide level. The animals weretreated as follows:

Group 1: Vehicle control

Group 2: Amaranth dried leaves powder (from example 12)—1 gm/kg

Group 3: Amaranth extract enriched with Amla extract (from example11)—200 mg/kg

After 30 minutes of feeding, blood was collected from all the animals inEDTA (ethylenediaminetetraaceticacid) coated tubes and plasma wasseparated immediately by centrifuging the tubes at 4000 rpm for 10minutes. The plasma was stored below −80° C. until analysis.

The nitric oxide (NO) content of the plasma is reported as μmol/L.

Baseline Nitric Nitric oxide S. oxide level level after 30 min No.Groups (μmol/L) (μmol/L) 1. Vehicle control 21.9 21.7 2. Amaranth driedleaves powder 20.8 22.6 (from example 12) 3. Amaranth extract enriched21.6 76.4 with Amla extract (from example 11)

EXAMPLE 26

Oral Toxicity Study in Rats.

This study was conducted as per OECD Guidelines (Organisation foreconomic co-operation and development) for Testing of chemicals(No.-408). 100 Wistar albino rats (50 males/50 females) were dividedinto four groups of 20 animals (10 males and 10 Females) in each and twogroups (Satellite groups) of 10 animals (5 males and 5 Females) in eachgroup. The animals were acclimatized for seven days before thecommencement of dosing. Three groups of 20 rats each (10 male and 10female) were administered with amaranth nitrate enriched extract (fromexample 8) at the dosage levels of 100 mg/kg body weight (low dose), 500mg/kg b.wt (body weight) (intermediate dose) and 1000 mg/kg body weight(high dose) respectively for seven days a week for 90 days with the helpof cannula attached with the syringe. Similarly, fourth group of 20 rats(10 male and 10 female rats) were orally administered with corn oil only(vehicle) for 90 days and was designated as control group.

Two additional satellite groups of 10 rats (5 male and 5 female) eachwere also kept and designated as ‘Satellite control’ and ‘Satellite highdose’ and were administered with corn oil (vehicle) and amaranth nitrateenriched extract (1000 mg/kg) respectively for 90 days. After terminalsacrifice of the test and control group animals, both Satellite groupanimals (Satellite control and Satellite high dose) were kept underobservation for an additional 28 days, to check the reversibility,persistence or delayed toxic effect, if any. The animals were observeddaily for behavior, appearance and toxicological signs and symptoms.Blood was collected from all the animals before terminal sacrifice fordetailed hematological and biochemical evaluation. Urine samples werealso collected from all animals at the termination of the experiment.Criteria used to evaluate compound related effects included; appearance,behavior, morbidity, mortality, body weights, Feed consumption,hematological and biochemical analysis, urine analysis, organ weights,necropsy and histopathology.

No treatment related toxic sign and symptoms were observed in low dose(100 mg/kg body weight), intermediate dose (500 mg/kg body weight), highdose (1000 mg/kg body weight.) and satellite high dose (1000 mg/kg bodyweight) group animals when compared to their respective control counterparts. Body weights of all the test and control group animals wererecorded weekly. Body weight gain of all the treatment groups andsatellite group animals (LD, ID, HD & satellite HD) was comparable totheir control counterparts. Feed consumption of the animals of low dose,intermediate dose, high dose and satellite high dose were comparable tothe control group and satellite control group animals.

There were no variations in the haematological parameters of animals oflow dose (100 mg/kg Body weight), intermediate dose (500 mg/kg bodyweight), high dose (1000 mg/kg body weight) and satellite high dose(1000 mg/kg body weight) groups when compared to the control groupanimals. Similarly, the biochemical parameters of animals of all thetreatment groups i.e. low dose, intermediate dose and high dose werecomparable to the biochemical parameters of the control group animals atthe terminal sacrifice. The biochemical parameters of satellite highdose group were also comparable to its satellite control counterparts.The heamatological as well as biochemical parameters of test groups werenot significantly different from respective control groups.

Urine samples were collected from all animals at the termination of theexperiment. No significant changes were noted in the urine parameters ofany of the dose group animals when compared to the control group.Animals of satellite groups were sacrificed after 28 days posttreatment. None of the animals died during the study in any of thetreated groups as well as the satellite groups. After completion ofdosing period of 90 days, all the groups (treatment and control) exceptboth the satellite groups were sacrificed and were examined for grosspathological findings. Organs of all the animals (treatment and control)were trimmed of any adherent fat tissue and their weights taken. Organweights of animals of all treatment groups were comparable to theirrespective control counter parts. There were no significanthistopathological changes in the animals of low dose group, intermediatedose group, high dose group and satellite high dose group when comparedto their control counterparts.

In this study, the repeated administration of amaranth nitrate enrichedextract for 90 days, by oral route, to wistar rats at the dosage levelof 1000 mg/kg body weight did not induce any observable toxic effects,when compared to its corresponding control group of animals.

EXAMPLE 27

Nitric Oxide Level in Human Subjects.

18 healthy human volunteers (age 35-55 years) were taken for the study.The subjects were put on standard diet (devoid of nitrate/nitritecontaining food) one week prior to the study. The subjects were dividedinto three groups comprising of six subjects in each group. The subjectswere fasted overnight and blood was collected to determine the baselinenitric oxide level. After blood collection, the subjects were treated asfollows:

Group 1: Vehicle control

Group 2: Amaranth fresh leaves as such—100 gm

Group 3: Amaranth fast dissolving tablet containing amaranth nitrateenriched extract (14.4 mg nitrate per tablet pepared as per example 21and extract used for making the tablet prepared as per example 7)

After 30 minutes of feeding, blood was collected from all the subjectsin EDTA (ethylene di amine tetra acetic acid) coated tubes and plasmawas separated immediately by centrifuging the tubes at 4000 rpm for 10minutes. The plasma was stored below −80° C. until analysis for nitricoxide content. The nitric oxide (NO) content of the plasma is reportedas μmol/L.

Baseline Nitric Nitric oxide S. oxide level level after 30 min No.Groups (μmol/L) (μmol/L) 1. Vehicle control 28.5 28.2 2. Amaranth freshleaves as such 29.2 30.1 3. Amaranth fast dissolving 29.1 78.9tablet(Fast melt prepared as per Example 21 and extract used for makingthe tablet prepared as per example 7)

EXAMPLE 28

Anti-hypertensive Activity in Human Subjects.

10 Subjects (6 males and 4 females) aged 40 to 60 years withpre-hypertension [SBP (systolic blood pressure), 130 to 140 mmHg and DBP(diastolic blood pressure), 85 to 90 mmHg) or hypertension (SBP(systolic blood pressure), 140 mmHg or higher and DBP (diastolic bloodpressure), 90 mmHg or higher) were enrolled in the study. Exclusioncriteria included pregnant and lactating women, people who had receivedtrace element supplements in the previous three months, people receivinggastric or diuretic treatments, patients with acute renal failure, orpeople with recent history of surgery or acute infections. All subjectswere informed of the purposes of the study, were free to ask questionsthroughout the study, and signed an informed consent form witnessed byone of the investigators.

Baseline blood Blood pressure Blood pressure pressure after 15 daysafter 30 days S. (SBP/DBP in (SBP/DBP in (SBP/DBP in No. Subjects mmHg)mmHg) mmHg) 1. Subject 1 138/87 135/84 130/82 2. Subject 2 142/90 138/88133/84 3. Subject 3 140/89 137/87 131/83 4. Subject 4 139/88 136/86132/83 5. Subject 5 143/91 138/88 134/85 6. Subject 6 141/90 136/87132/84 7. Subject 7 139/90 135/83 129/81 8. Subject 8 138/86 134/83128/80 9. Subject 9 142/90 137/87 131/84 10. Subject 10 142/90 136/86132/83

The patients were provided with a bottle containing 60 fast melt mouthdissolving tablets (from Example 21) and instructed to take one tablettwice daily at 12 hour interval for 30 days. Eating habits were notrestricted during the treatment. However, to avoid possible interferencewith the effects of PG, the consumption of vitamin supplements andfunctional foods was prohibited during the trial. Each subject's BP(blood pressure) was measured at the beginning, 15 days and end of thetrial. Their BP (blood pressure) was measured using a digital BPanalyzer and recorded as the average of two measurements. Measurementswere always made at the same place and time after 15 min of rest.

EXAMPLE 29

Swimming Endurance Test in Mice.

For swimming endurance study, prior to experiment, each mouse was testedfor feasibility testing. All mice in swimming groups could swim. Themice were divided into 3 groups comprising of 6 animals in each group.Following treatment was given to the respective group of mice:

Group 1: Vehicle control

Group 2: Amaranth dried leaves powder (from example 12)—1 gm/kg

Group 3: Amaranth nitrate enriched extract (from example 8)—100 mg/kg

Treatment was given for four weeks.

All mice were subjected to swim in a water pool (48×28×24 inch), filled19 inch deep of water maintained at 25° C. During swimming testing, eachmouse was fixed four paper clips with a plastic string on the tail. Thepaper clips and plastic string weighted about 1.81 grams. Bearing thisweight, all mice were forced to swim very hard. All mice were subjectedto swim until exhaustion (cease movement of limbs and float). Themaximum time swimming until mice exhausted was used as the index ofswimming endurance capacity. The test was conducted in the beginning andthen weekly till four weeks. Blood was also collected in the beginningand after each week to determine the nitric oxide level in plasma.

Swimming endurance (seconds) S. Base- 1 2 3 4 No. Groups line Week WeeksWeeks Weeks 1. Vehicle control 130 126 128 130 127 2. Amaranth dried 132146 168 172 179 leaves powder (from example 12) 3. Amaranth nitrate 128155 171 185 193 enriched extract (from example 8)

Nitric Oxide Level

Nitric oxide level (μmol/L) S. Base- 1 2 3 4 No. Groups line Week WeeksWeeks Weeks 1. Vehicle control 17.6 17.3 18.2 18.0 17.9 2. Amaranthdried 17.9 18.1 18.3 17.7 17.9 leaves powder (from example 12) 3.Amaranth nitrate 17.7 32.1 46.5 51.3 56.9 enriched extract (from example8)

EXAMPLE 30

Running Endurance Test in Mice.

For running endurance study, individual mouse was trained for running ona Rota-Rod apparatus (treadmill) for three days prior to running study.During training, the speed of the Rota-rod was increased incrementallyto make animals exercise more intensely and running for at least oneminute. The mice were divided into 3 groups comprising of 6 animals ineach group. Following treatment was given to the respective group ofmice:

Group 1: Vehicle control

Group 2: Amaranth dried leaves powder (from example 12)—1 gm/kg

Group 3: Amaranth nitrate enriched extract (from example 9)—100 mg/kg

Treatment was given for four weeks.

The Rota-Rod treadmills had five testing zones, each zone had a photobean that sensed when the animal dropped off the treadmills which wasconnected with computer to calculate running time on the treadmillsautomatically. The speed of the Rota-rod was set to 35 RPM (rotation perminute). The maximum time running until exhaustion (drops from theRota-rod) was used as the index of running endurance capacity. Themaximum time of running until exhaustion in each mouse was recorded. Thetest was conducted in the beginning and then weekly till four weeks.

Running endurance (seconds) S. Base- 1 2 3 4 No. Groups line Week WeeksWeeks Weeks 1. Vehicle control 452 445 454 450 448 2. Amaranth dried 436451 472 481 486 leaves powder (from example 12) 3. Amaranth nitrate 441468 489 497 510 enriched extract (from example 9)

EXAMPLE 31

Sexual Performance Study in Rats.

Twelve male wistar rats (200-250 gm) were randomized into 2 groupscomprising of 6 animals each. The animals were treated for 30 days withvehicle/extract and various test parameters were evaluated.

Group I (vehicle only) served as control.

Group II Amaranth extract enriched with amla extract (from example11)—100 mg/Kg (p.o.) daily

Orientation Behavior Analysis

The analysis of orientation activity was carried out and analyzed inthree segments. Orientation behavior of male rats was determined usingfollowing method of scoring: Orientation towards female—(1 for everysniffing and 2 for every licking)

Orientation towards self—(1 for non-genital grooming and 2 for genitalgrooming)

Orientation towards environment—(1 for exploration, 2 for rearing and 3for climbing)

Rats were observed daily for their orientation activity. The cumulativescore after 0, 15, 30 days of the treatment were recorded.

Sexual Behavior Analysis

Male rat was placed in the observation glass chambers in order toacclimatize it with the cage environment. Sexually receptive female ratwas then allowed to enter the test cage silently from a side door insidethe cage. The behavioral observations were carried out taking intoaccount the following parameters.

Mounting Behavior—It was determined and characterized by followingparameters.

(A) Mount frequency—average number of mount during 30 min observation.

(B) Mount latency—The leg time from the introduction of female in thecage to first mount.

Intromission Behavior—It was evaluated as follows.

(A) Intromission frequency—average number of Intromission during 30 minobservation.

(B) Intromission latency—Intromission latency (IL) was considered as thetime for first intromission after introduction of female in the cage.

Penile Erection Index

Penile Erection (PE) was determined when the rats bent down to licktheir erect penis during the observation period. Penile erection index(PI) was determined by multiplying the percentage of rats exhibiting atleast one episode of penile erection during 30-min observation periodwith the mean number of penile erections.

PI=% of rats exhibiting penile erection×Mean number of erections.

TABLE Effect of enriched amaranth extract on orientation activities inmale rats Parameters for sexual behavior 15 days of 30 days of analysisGroups 0 days treatment treatment Mean activity Vehicle control 7.5 7.67.5 Score Towards Amaranth 7.4 11.2 16.3 Female (Licking & extractenriched Anogenital with Amla smelling) extract Mean activity Vehiclecontrol 13.2 13.3 13.6 Score Towards Amaranth 13.3 16.5 22.2 Environmentextract enriched (Exploration, with Amla Rearing and extract Climbing)Mean activity Vehicle control 8.1 8.0 8.1 Score Towards Amaranth 7.912.6 15.3 Self (Nongenital extract enriched grooming and with AmlaGenital grooming) extract

TABLE Effect of enriched amaranth extract on sexual performanceparameters in male rats Parameters for 30 days sexual behavior 15 daysof of analysis Groups 0 days treatment treatment Mount latency Vehiclecontrol 194.3 195.6 195.2 (time in seconds) Amaranth extract 193.5 160.2124.3 enriched with Amla extract Mount frequency Vehicle control 3.3 3.53.8 Amaranth extract 3.4 10.5 12.4 enriched with Amla extractIntromission Vehicle control 305.2 304.3 302.6 latency (time in Amaranthextract 309.5 275.3 254.4 seconds) enriched with Amla extractIntromission Vehicle control 1.6 1.7 1.7 frequency Amaranth extract 1.54.2 6.3 enriched with Amla extract Penile Erection Vehicle control 23.322.7 24.5 Index (PI) Amaranth extract 21.5 45.3 62.6 enriched with Amlaextract

EXAMPLE 32

Nitrate (NO₃) Nitrite (NO₂) and Nitric Oxide (NO) Content in Plasma ofHuman Subjects

32 Healthy human volunteers (age 35-55 years) were taken for the study.The subjects were put on standard diet one week prior to the study. Thesubjects were divided into 8 groups comprising of four subjects in eachgroup. The subjects were fasted overnight and the blood was collected todetermine the baseline NO₃ and NO₂ levels. After baseline bloodcollection, the subjects were treated as follows:

Group 1: Vehicle Control.

Group 2: Amaranth leaves fresh juice as such—100 ml (100 mg Nitrate/100ml of juice) (Prepared as per example No-13)

Group 3: Amaranth fast dissolving tablet containing Methanol extract ofAmaranth fresh leaves (7.5 mg nitrate per tablet) (Tablet prepared asper example No. 22 and extract used for preparing the tablet is as perexample 1)

Group 4: Amaranth fast dissolving tablet containing Methanol extract ofAmaranth dried leaves (8.25 mg nitrate per tablet) (Tablet prepared asper example No. 22 and extract used for preparing the tablet is as perexample 3)

Group 5: Amaranth fast dissolving tablet containing water extract ofAmaranth fresh leaves (8 mg nitrate per tablet) (Tablet prepared as perexample No 22 and extract used for preparing the tablet is as perexample 2)

Group 6: Amaranth fast dissolving tablet containing water extract ofAmaranth dried leaves (8.5 mg nitrate per tablet) (Tablet prepared asper example No 22 and extract used for preparing the tablet is as perexample 4)

Group 7: Amaranth fast dissolving tablet containing Pectinase treatedwater extract of Amaranth fresh leaves (10 mg nitrate per tablet)(Tablet prepared as per example No 22 and extract used for preparing thetablet is as per example 5)

Group 8: Amaranth fast dissolving tablet containing amaranth nitrateenriched extract (90 mg nitrate per tablet) (Tablet prepared as perexample No 22 and extract used for preparing the tablet is as perexample 7)

All subjects were allowed to consume the water/Juice orally (Group No-1to 2). Amaranth Tablet were kept in oral cavity and allow to dissolve(Group No-3 to 8).

After 1 hour, 2 hour and 3 hour from feeding of tablet/juice/vehicle,blood was drawn from all the subjects in EDTA coated tubes. Plasma wasseparated immediately by centrifuging the tubes at 5000 rpm for 15 min.The plasma samples were stored below −80 degree Celsius until analysis.The plasma thus obtained was deproteinized using Acetonitrile and thesupernatant liquid was used for nitrate/nitrite determination usingHPLC. NO₃ was analyzed by HPLC on a BETASIL C-18 column (250×4.6 mm)using Tetra butyl ammonium hydroxide in De-ionized water, acetonitrileand methanol as mobile phase and

Photo diode array detection at 222 nm. NO₂ was analysed at 520 nm usingthe same conditions. The NO₃ and NO₂ levels in plasma were reported asμmol/L.

TABLE Nitrate (NO₃) content in plasma Baseline NO₃ NO₃ NO₃ NO₃ levelslevels levels SI levels after 1 hour after 2 hour after 3 hour NO Groups(μmol/l) (μmol/l) (μmol/l) (μmol/l) 1. Vehicle Control 28.92 28.95 27.8828.81 2. Amaranth fresh juice as 28.55 30.11 29.45 28.56 such 3.Amaranth fast dissolving 27.88 30.12 29.88 28.65 tablet containingmethanol extract of fresh leaves 4. Amaranth fast dissolving 26.55 30.1930.02 28.89 tablet containing water extract of fresh leaves 5. Amaranthfast dissolving 29.52 33.20 30.25 29.56 tablet containing methanolextract of dried leaves. 6. Amaranth fast dissolving 29.10 32.8 31.0229.77 tablet containing water extract of dried leaves 7. Amaranth fastdissolving 29.11 32.92 31.95 30.10 tablet containing pectinase treatedwater extract of fresh leaves 8. Amaranth fast dissolving 28.88 174.40110.22 70.89 tablet containing enriched extract of nitrate

TABLE Nitrite (NO₂) content in plasma Base line NO₂ NO₂ NO₂ NO₂ levelslevels levels SI levels after 1 hour after 2 hour after 3 hour No Groups(μmol/l) (μmol/l) (μmol/l) (μmol/l) 1. Vehicle Control 0.19 0.20 0.230.18 2. Amaranth fresh juice as 0.21 0.25 0.24 0.20 such 3. Amaranthfast dissolving 0.20 0.27 0.26 0.24 tablet containing methanol extractof fresh leaves 4. Amaranth fast dissolving 0.21 0.29 0.27 0.25 tabletcontaining water extract of fresh leaves 5. Amaranth fast dissolving0.19 0.28 0.27 0.24 tablet containing methanol extract of dried leaves.6. Amaranth fast dissolving 0.21 0.31 0.35 0.30 tablet containing waterextract of dried leaves 7. Amaranth fast dissolving 0.19 0.30 0.28 0.25tablet containing pectinase treated water extract of fresh leaves 8.Amaranth fast dissolving 0.20 1.20 0.98 0.60 tablet containing enrichedextract of nitrate

TABLE Total nitric oxide (NO) content in plasma Base line NO NO NO NOlevels levels levels SI levels after 1 hour after 2 hour after 3 hour NOGroups (μmol/l) (μmol/l) (μmol/l) (μmol/l) 1. Vehicle Control 29.1129.15 28.11 28.99 2. Amaranth fresh juice as 28.76 30.36 29.69 28.76such 3. Amaranth fast dissolving 28.08 30.39 30.14 28.89 tabletcontaining methanol extract of fresh leaves 4. Amaranth fast dissolving26.76 30.48 30.29 29.14 tablet containing water extract of fresh leaves5. Amaranth fast dissolving 29.71 33.48 30.52 29.80 tablet containingmethanol extract of dried leaves. 6. Amaranth fast dissolving 29.3133.11 31.37 30.07 tablet containing water extract of dried leaves 7.Amaranth fast dissolving 29.30 33.22 32.23 30.35 tablet containingpectinase treated water extract of fresh leaves 8. Amaranth fastdissolving 29.08 175.60 111.2 71.49 tablet containing enriched extractof nitrate

Amaranth fresh juice consumption as such slightly increased the NO₃content after one hour. Methanol extract from amaranth fresh leaves anddried leaves also showed a slight increase in NO₃ content. Tablet madewith pectinase treated water extract of fresh leaves of Amaranth shown alittle more increase than the other tablets consumptions. But afterconsumption of fast dissolving tablet containing Amaranth extractenriched with nitrate, NO₃ level increased to 6 times the baseline valueafter 1 hour and the increase from base line is maintained up to 3 hour.There was no significant change in NO₃ level after administering thevehicle.

In case of NO₂ also, the fast dissolving tablet containing Amaranthextract enriched with nitrate, increased the NO₂ level more than 6 timesand the higher levels maintained for 3 hours. Amaranth fresh juiceconsumption as such slightly increased the NO₂ content after one hour.Methanol extract from amaranth fresh leaves and dried leaves also showeda slight increase in NO₂ content. Here also vehicle did not influenceNO₂ level in the subjects.

Overall conclusion is that the increased levels of NO₃ and NO₂ increaseobserved in Amaranth fast dissolving tablet was more when compared withlevels of nitrate and nitrite in Amaranth fresh juice, and methanolextract from fresh of dried leaves. The increase in total nitric oxide(NO) content of “Amaranth fast dissolving tablet containing enrichedextract of nitrate” was 6 times from base line and it remained increasedfor 3 hours. Total NO is determined from the sum of total NO₃ and NO₂.(ie. Level of NO₃ and NO₂ can be interpreted as a measure of NO level inhuman body) So an increase in NO₃ and NO₂ level is interpreted as anincrease in NO level. NO levels was recorded as the sum of the two majormetabolites of NO, nitrite and nitrate.

EXAMPLE 33

Nitrate (NO₃), Nitrite (NO₂) and Nitric Oxide (NO) Content in Saliva ofHuman Subjects

32 Healthy human volunteers (age 35-55 years) were taken for the study.The subjects were put on controlled diet, one week prior to the study.The subjects were divided into eight groups comprising of four subjectsin each group. The subjects were fasted overnight and saliva wascollected to determine the base line NO₃ and NO₂ levels. After salivacollection, the subjects were treated as follows:

Group 1: Vehicle Control.

Group 2: Amaranth leaves fresh juice as such—100 ml (100 mg Nitrate/100ml of juice) (Prepared as per example No. 13).

Group 3: Amaranth fast dissolving tablet containing Methanol extract ofAmaranth fresh leaves (7.5 mg nitrate per tablet) (Tablet prepared asper example No 22 and extract used for preparing the tablet is as perexample 1).

Group 4: Amaranth fast dissolving tablet containing Methanol extract ofAmaranth dried leaves (8.25 mg nitrate per tablet) (Tablet prepared asper example No. 22 and extract used for preparing the tablet is as perexample 3).

Group 5: Amaranth fast dissolving tablet containing water extract ofAmaranth fresh leaves (8 mg nitrate per tablet) (Tablet prepared as perexample No 22 and extract used for preparing the tablet is as perexample 2).

Group 6: Amaranth fast dissolving tablet containing water extract ofAmaranth dried leaves (8.5 mg nitrate per tablet) (Tablet prepared asper example No 22 and extract used for preparing the tablet is as perexample 4)

Group 7: Amaranth fast dissolving tablet containing Pectinase treatedwater extract of Amaranth fresh leaves (10 mg nitrate per tablet)(Tablet prepared as per example No 22 and extract used for preparing thetablet is as per example 5)

Group 8: Amaranth fast dissolving tablet containing amaranth nitrateenriched extract (90 mg nitrate per tablet) (Tablet prepared as perexample No 22 and extract used for preparing the tablet is as perexample 7)

All subjects were allowed to consume the water/Juice orally (Group No-1to 2). Amaranth Tablet were kept in oral cavity and allow to dissolve(Group No-3 to 8)

After each of the time point 1 hour, 2 hour and 3 hour from feeding,saliva was collected from all subjects in 5.0 ml PTFE tubes. This salivawas stored below −80 degree Celsius until analysis for NO₃ and NO₂content. The saliva thus obtained was deprotenized using Acetonitrileand the supernatant liquid was used for NO₃ and NO₂ determination usingHPLC. NO₃ was analyzed by HPLC on a BETASIL C-18 column (250×4.6 mm)using Tetra butyl ammonium hydroxide in De-ionized water, acetonitrileand methanol as mobile phase and Photo diode array detection at 222 nm.NO₂ was analyzed at 520 nm using the same conditions. The Nitrate andNitrite of the saliva are reported as μmol/L.

TABLE Nitrate (NO₃) content in saliva Base line NO₃ NO₃ NO₃ NO₃ levelslevels levels SI levels after 1 hour after 2 hour after 3 hour NO Groups(μmol/l) (μmol/l) (μmol/l) (μmol/l) 1. Vehicle Control 292.54 295.66295.95 290.57 2. Amaranth fresh juice as such 295.56 298.25 297.20295.57 3. Amaranth fast dissolving 296.55 299.35 299.10 297.01 tabletcontaining methanol extract of fresh leaves 4. Amaranth fast dissolving298.82 302.72 300.2 299.84 tablet containing water extract of freshleaves 5. Amaranth fast dissolving 295.02 299.06 298.35 296.77 tabletcontaining methanol extract of dried leaves 6. Amaranth fast dissolving298.25 302.35 301.5 299.56 tablet containing water extract of driedleaves 7. Amaranth fast dissolving 296.50 300.78 298.8 297.23 tabletcontaining pectinase treated water extract of fresh leaves 8. Amaranthfast dissolving 297.80 2062.25 2120.2 1895.51 tablet containing enrichedextract of nitrate

TABLE Nitrite (NO₂) content in saliva Base line NO₂ NO₂ NO₂ NO₂ levelslevels levels SI levels after 1 hour after 2 hour after 3 hour NO Groups(μmol/l) (μmol/l) (μmol/l) (μmol/l) 1. Vehicle Control 112.54 113.55112.95 115.39 2. Amaranth fresh juice as 115.26 122.57 120.68 116.56such 3. Amaranth fast dissolving 113.50 121.00 120.65 119.82 tabletcontaining methanol extract of fresh leaves 4. Amaranth fast dissolving118.82 126.42 124.66 118.56 tablet containing water extract of freshleaves 5. Amaranth fast dissolving 114.93 122.63 120.44 119.64 tabletcontaining methanol extract of dried leaves. 6. Amaranth fast dissolving115.65 123.45 120.23 117.38 tablet containing water extract of driedleaves 7. Amaranth fast dissolving 118.56 126.46 125.56 120.25 tabletcontaining pectinase treated water extract of fresh leaves 8. Amaranthfast dissolving 115.78 1075.20 1005.20 782.50 tablet containing enrichedextract of nitrate

TABLE Total nitric oxide (NO)content in saliva Base line NO NO NO NOlevels levels levels SI levels after 1 hour after 2 hour after 3 hour NOGroups (μmol/l) (μmol/l) (μmol/l) (μmol/l) 1. Vehicle Control 405.08409.21 408.9 405.96 2. Amaranth fresh juice as 410.82 420.82 417.88412.13 such 3. Amaranth fast dissolving 410.05 420.35 419.75 416.83tablet containing methanol extract of fresh leaves 4. Amaranth fastdissolving 417.64 429.14 424.86 418.80 tablet containing water extractof fresh leaves 5. Amaranth fast dissolving 409.95 421.69 418.79 416.41tablet containing methanol extract of dried leaves. 6. Amaranth fastdissolving 413.90 425.80 421.73 416.94 tablet containing water extractof dried leaves 7. Amaranth fast dissolving 415.06 427.24 424.36 417.48tablet containing pectinase treated water extract of fresh leaves 8.Amaranth fast dissolving 413.58 3137.45 3125.40 2678.01 tabletcontaining enriched extract of nitrate

Amaranth fresh juice consumption as such slightly increased the NO₃content after one hour. Methanol extract from amaranth fresh leaves anddried leaves also showed a slight increase in NO₃ content. Tablet madewith pectinase treated water extract of fresh leaves of Amaranth shown alittle more increase than the other tablets consumptions. But afterconsumption of fast dissolving tablet containing Amaranth extractenriched with nitrate, NO₃ level increased to 6.9 fold the baselinevalue after 1 hour and the increase in NO₃ was maintained for 3 hours.There was no significant change in NO₃ level after administering thevehicle.

In case of NO₂ also, the fast dissolving tablet containing Amaranthextract enriched with nitrate, increased the NO₂ level more than 9.2fold and the increase in NO₂ was maintained for 3 hours. Amaranth freshjuice consumption as such slightly increased the NO₂ content after onehour. Methanol extract from amaranth fresh leaves and dried leaves alsoshowed a slight increase in NO₂ content. Here also vehicle did notinfluenced NO₂ level in the subjects.

Overall conclusion is that the total NO₃ and NO₂ level increase wasobserved in Amaranth fast dissolving tablet was more comparedwith—Amaranth fresh juice, Methanol extract from fresh and dried leaves.The increase in total NO content of “Amaranth fast dissolving tabletcontaining enriched extract of nitrate” is 7.5 fold from base line andit remained increased for 3 hours. Total NO was interpreted as the sumof total NO₃ and NO₂. Level of NO₃ and NO₂ was used to interpret thelevel of NO level in human body. So an increase in NO₃ and NO₂ level wasinterpreted as an increase in NO level. NO levels was recorded as thesum of the two major metabolites of NO, namely, nitrite and nitrate.(Journal of Medical sciences (2010):3 (3): 153-159, Plasma Endothelin—1,Homocysteine, and Oxide Levels in a Multiethnic Hypertensive Cohort fromthe United Arab Emirates.)

EXAMPLE 34

Nitrate (NO₃) Nitrite (NO₂) and Nitric Oxide (NO) Content in Saliva ofHuman Subjects

18 Healthy human volunteers (age 35-55 years) were taken for the study.The subjects were put on controlled diet, one week prior to the study.The subjects were divided into three groups comprising of six subjectsin each group. The subjects were fasted overnight and saliva wascollected to determine the base line NO₃ and NO₂ levels. After salivacollection, the subjects were treated as follows:

Group 1: Vehicle control. (Water)

Group 2: Amaranth leaves fresh juice as such—100 ml (100 mg Nitrate/100ml of juice) (Prepared as per example No-13)

Group 3: Amaranth extract with enriched nitrate (18% nitrate prepared asper example 7) is mixed with water. (560 mg of extract in 300 ml water)

All subjects were allowed to consume the water/Juice orally (Group No-1to 2). Amaranth extract enriched with nitrate mixed with water wasconsumed directly in the oral cavity (Group No-3)

After each of the time point 1 hour, 2 hour and 3 hour from feeding,saliva was collected from all subjects in 5.0 ml PTFE tubes. This salivawas stored below −80 degree Celsius until analysis for NO₃ and NO₂content. The saliva thus obtained was deprotenized using Acetonitrileand the supernatant liquid was used for NO₃ and NO₂ determination usingHPLC. NO₃ was analyzed by HPLC on a BETASIL C-18 column (250×4.6 mm)using Tetra butyl ammonium hydroxide in De-ionized water, acetonitrileand methanol as mobile phase and Photo diode array detection at 222 nm.NO₂ was analyzed at 520 nm using the same conditions. The Nitrate andNitrite of the saliva were reported as μmol/L.

TABLE Nitrate (NO₃) content in saliva Base line NO₃ NO₃ NO₃ NO₃ levelslevels levels SI levels after 1 hour after 2 hour after 3 hour NO Groups(μmol/l) (μmol/l) (μmol/l) (μmol/l) 1. Vehicle Control 295.65 296.55293.78 296.58 2. Amaranth fresh juice 296.23 300.02 297.02 296.33 assuch 3. Amaranth extract 295.68 1961.51 1901.27 789.56 with enrichednitrate mixed with water

TABLE Nitrite (NO₂) content in saliva Base line NO₂ NO₂ NO₂ NO₂ levelslevels levels SI levels after 1 hour after 2 hour after 3 hour NO Groups(μmol/l) (μmol/l) (μmol/l) (μmol/l) 1. Vehicle Control 110.56 112.56110.94 113.24 2. Amaranth fresh juice 113.54 120.56 119.85 113.55 assuch 3. Amaranth extract 115.60 1060.45 1055.33 814.34 with enrichednitrate mixed with water

TABLE Total nitric oxide (NO) content in Saliva Base line NO NO NO NOlevels levels levels SI levels after 1 hour after 2 hour after 3 hour NOGroups (μmol/l) (μmol/l) (μmol/l) (μmol/l) 1. Vehicle Control 406.21409.11 404.72 409.82 2. Amaranth fresh juice 409.77 420.58 416.87 409.88as such 3. Amaranth extract 411.28 3021.96 2956.60 1603.90 with enrichednitrate mixed with water

Amaranth fresh juice consumption as such slightly increased the NO₃content after one hour. But after consumption of Amaranth extract withenriched nitrate mixed with water, NO₃ level increased to 6.6 fold thebaseline value after 1 hour and remained increased for 3 hours. Therewas no significant change in NO₃ level after administering the vehicle.

In case of NO₂ also, Amaranth extract with enriched nitrate mixed withwater increased the NO₂ level 9.1 fold and remained increased for 3hours. Amaranth fresh juice consumption as such slightly increased theNO₂ content after one hour.

Overall conclusion is that the total NO₃ and NO₂ level increase observedby administering Amaranth enriched extract mixed with water was morewhen compared with administering Amaranth fresh juice. The increase intotal NO content of “Amaranth enriched extract mixed with water” was 7.3times from baseline and it remained increased for 3 hours.

EXAMPLE 35

Nitrate (NO₃), Nitrite (NO₂) and Nitric Oxide (NO) Content in Plasma andSaliva of Human Subjects After Administering Extract of AmaranthusBlitum and Amaranthus Viridus with Enriched Nitrate.

Nine healthy human volunteers (age 35-55 years) were taken for thestudy. The subjects were put on standard diet one week prior to thestudy. The subjects were divided into three groups comprising of threesubjects in each group. The subjects were fasted overnight and the bloodand saliva was collected to determine the baseline NO₃ and NO₂ levels.After baseline blood and saliva collection, the subjects were treated asfollows:

Group 1: Vehicle Control.

Group 2: Amaranth fast dissolving tablet containing extract ofAmaranthus blitum with enriched nitrate (91.5 mg nitrate per tablet)(Tablet prepared as per example No 22 and extract used for preparing thetablet is as per example 15).

Group 3: Amaranth fast dissolving tablet containing extract ofAmaranthus viridus with enriched nitrate (90.5 mg nitrate per tablet)(Tablet prepared as per example No 22 and extract used for preparing thetablet is as per example 15).

All subjects were allowed to consume the water orally (Group No. 1).Amaranth Tablets were kept in oral cavity and allow to dissolve (GroupNo. 2 to 3).

After 1 hour, 2 hour and 3 hour from feeding of tablet/vehicle, bloodwas drawn from all the subjects in EDTA coated tubes and saliva wascollected from the same subjects in 5.0 ml PTFE tubes. Plasma wasseparated immediately by centrifuging the tubes at 5000 rpm for 15 min.The plasma samples and saliva were stored below −80 degree Celsius untilanalysis. The plasma and saliva were deproteinized using Acetonitrileand the supernatant liquid was used for nitrate/nitrite determinationusing HPLC. NO₃ was analyzed by HPLC on a BETASIL C-18 column (250×4.6mm) using Tetra butyl ammonium hydroxide in De-ionized water,acetonitrile and methanol as mobile phase and photo diode arraydetection at 222 nm. NO₂ was analysed at 520 nm using the sameconditions. The NO₃ and NO₂ levels in plasma were reported as μmol/L.

Nitrate (NO₃), Nitrite (NO₂) and Nitric Oxide (NO) Level in Plasma

TABLE Nitrate (NO₃) content in plasma Baseline NO₃ NO₃ NO₃ NO₃ levelslevels levels SI levels after 1 hour after 2 hour after 3 hour NO Groups(μmol/l) (μmol/l) (μmol/l) (μmol/l) 1. Vehicle Control 28.92 28.95 27.8828.81 2. Fast dissolving tablet 28.41 172.50 109.43 70.5 containingextract of Amaranthus blitum with enriched nitrate. 3. Fast dissolvingtablet 27.65 168.92 110.22 70.89 containing extract of Amaranthusviridus with enriched nitrate.

TABLE Nitrite (NO₂) content in plasma Base line NO₂ NO₂ NO₂ NO₂ levelslevels levels SI levels after 1 hour after 2 hour after 3 hour No Groups(μmol/l) (μmol/l) (μmol/l) (μmol/l) 1. Vehicle Control 0.19 0.20 0.230.18 2. Fast dissolving tablet 0.19 1.15 0.94 0.59 containing extract ofAmaranthus blitum with enriched nitrate. 3. Fast dissolving tablet 0.211.27 0.97 0.65 containing extract of Amaranthus viridus with enrichednitrate.

TABLE Total nitric oxide (NO) content in plasma Base line NO NO NO NOlevels levels levels SI levels after 1 hour after 2 hour after 3 hour NOGroups (μmol/l) (μmol/l) (μmol/l) (μmol/l) 1. Vehicle Control 29.1129.15 28.11 28.99 2. Fast dissolving tablet 28.6 173.65 110.37 71.09containing extract of Amaranthus blitum with enriched nitrate 3. Fastdissolving tablet 27.86 170.19 111.19 71.54 containing extract ofAmaranthus viridus with enriched nitrate.

After consumption of fast dissolving tablet containing extract ofAmaranthus blitum and Amaranthus viridus with enriched nitrate, NO₃level increased to 6 fold the baseline value after 1 hour and theincrease from base line was maintained up to 3 hour. There was nosignificant change in NO₃ level after administering the vehicle.

In case of NO₂ also, the fast dissolving tablet containing extract ofAmaranthus blitum and Amaranthus viridus with enriched nitrate,increased the NO₂ level more than 6 times and the higher levelsmaintained for 3 hours. Here also vehicle did not influence NO₂ level inthe subjects.

The increase in total nitric oxide (NO) content of “Amaranth fastdissolving tablet containing extract of Amaranthus blitum and Amaranthusviridus with enriched nitrate” was 6 times from base line and itremained increased for 3 hours. Total NO was determined from the sum oftotal NO₃ and NO₂. (i.e. Level of NO₃ and NO₂ can be interpreted as ameasure of NO level in human body). So an increase in NO₃ and NO₂ levelwas interpreted as an increase in NO level.

Nitrate (NO₃), Nitrite (NO₂) and Nitric Oxide (NO) Level in Saliva

TABLE Nitrate (NO₃) content in saliva Base line NO₃ NO₃ NO₃ NO₃ levelslevels levels SI levels after 1 hour after 2 hour after 3 hour NO Groups(μmol/l) (μmol/l) (μmol/l) (μmol/l) 1. Vehicle Control 292.54 295.66295.95 290.57 2. Fast dissolving tablet 297.10 2058.9 2184.2 1796.51containing extract of Amaranthus blitum with enriched nitrate 3. Fastdissolving tablet 296.7 2047.52 2147.2 1801.12 containing extract ofAmaranthus viridus with enriched nitrate

TABLE Nitrite (NO₂) content in saliva Base line NO₂ NO₂ NO₂ NO₂ levelslevels levels SI levels after 1 hour after 2 hour after 3 hour NO Groups(μmol/l) (μmol/l) (μmol/l) (μmol/l) 1 Vehicle Control 112.54 113.55112.95 115.39 2 Fast dissolving tablet 115.75 1064.9 1021.1 796.70containing extract of Amaranthus blitum with enriched nitrate 3 Fastdissolving tablet 113.58 1033.57 1001.4 701.3 containing extract ofAmaranthus viridus with enriched nitrate

TABLE Total nitric oxide (NO) content in saliva Base line NO NO NO NOlevels levels levels SI levels after 1 hour after 2 hour after 3 hour NOGroups (μmol/l) (μmol/l) (μmol/l) (μmol/l) 1. Vehicle Control 405.08409.21 408.9 405.96 2. Fast dissolving tablet 412.85 3123.8 3205.32593.21 containing extract of Amaranthus blitum with enriched nitrate 3.Fast dissolving tablet 410.28 3081.09 3148.6 2502.42 containing extractof Amaranthus viridus with enriched nitrate

After consumption of fast dissolving tablet containing extract ofAmaranthus blitum and Amaranthus viridus with enriched nitrate, NO₃level increased to 6.9 fold the baseline value after 1 hour and theincrease in NO₃ was maintained for 3 hours. There was no significantchange in NO₃ level after administering the vehicle.

In case of NO₂ also, the fast dissolving tablet containing extract ofAmaranthus blitum and Amaranthus viridus with enriched nitrate,increased the NO₂ level more than 9.2 fold and the increase in NO₂ wasmaintained for 3 hours. Here also vehicle did not influenced NO₂ levelin the subjects.

The increase in total NO content of “Amaranth fast dissolving tabletcontaining extract of Amaranthus blitum and Amaranthus viridus withenriched nitrate” was 7.5 fold from base line and it remained increasedfor 3 hours. Total NO was interpreted as the sum of total NO₃ and NO₂.(Level of NO₃ and NO₂ is used to interpret the level of NO level inhuman body). So an increase in NO₃ and NO₂ level was interpreted as anincrease in NO level.

It will be readily understood by the skilled artisan that numerousalterations may be made to the examples and instructions given herein.These and other objects and features of disclosed embodiments will bemade apparent from the following examples. The following examples asdescribed are not intended to be construed as limiting the scope of thedisclosure.

I claim:
 1. A tablet or capsule for lowering blood pressure in a humanin need thereof consisting essentially of therapeutically effectiveamounts of amla extract, Amaranth extract, green tea extract,pomegranate extract, turmeric extract and star anise extract.
 2. Atablet or capsule for increasing physical endurance in a human in needthereof consisting essentially of therapeutically effective amounts ofamla extract, Amaranth extract, green tea extract, pomegranate extract,turmeric extract and star anise extract.
 3. A tablet or capsule forincreasing swimming endurance in a human in need thereof consistingessentially of therapeutically effective amounts of amla extract,Amaranth extract, green tea extract, pomegranate extract, turmericextract and star anise extract.
 4. A tablet or capsule for increasingrunning endurance in a human in need thereof consisting essentially oftherapeutically effective amounts of amla extract, Amaranth extract,green tea extract, pomegranate extract, turmeric extract and star aniseextract.